WO2021207085A1 - Synthetic elaboration of native dna by rass (sendr) - Google Patents

Synthetic elaboration of native dna by rass (sendr) Download PDF

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WO2021207085A1
WO2021207085A1 PCT/US2021/025799 US2021025799W WO2021207085A1 WO 2021207085 A1 WO2021207085 A1 WO 2021207085A1 US 2021025799 W US2021025799 W US 2021025799W WO 2021207085 A1 WO2021207085 A1 WO 2021207085A1
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oligonucleotide
dna
triphosphate
modified
resin
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PCT/US2021/025799
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French (fr)
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Philip Dawson
Dillon FLOOD
Kyle KNOUSE
Phil BARAN
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The Scripps Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/02—Phosphorylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the present disclosure relates to the fields of chemical biology and therapeutics, and more specifically methods of modifying oligonucleotides.
  • Oligonucleotide conjugates are often used in the fields of chemical biology, biophysics, and diagnostics. Indeed, oligonucleotide conjugate-based technology has provided the basis for many modem technological advances. For example, DNA-PAINT conjugates enable transforming super resolution microscopy, and TaqMan PCR probes have revolutionized precision diagnostics.
  • Hybridization probes operate through an extraordinarily molecular recognition ability that a single strand of DNA displays towards its complimentary sequence.
  • the ability for DNA to take on defined inter and intramolecular conformations allows for another dimension of selectivity.
  • Visualizing these molecular interactions usually requires the incorporation of a fluorophore or radioactive moiety. It is this foundation that allows for ubiquitous biochemical and diagnostic techniques such as Southern and Northern Blotting, molecular beacons, and TaqMan qPCR.
  • DNA-PAINT and TaqMan PCR require custom DNA oligomer conjugates that are precisely functionalized and homogeneous. Chemical synthesis of the requisite oligonucleotide conjugates is costly and time consuming. This can make diagnosis of diseases and laboratory experiments cost prohibitive. For example, a custom TaqMan PCR probe must be bought for every diagnostic test or experiment, making it enormous expensive. Furthermore, as oligonucleotide tagging becomes increasingly popular, multiplexed experiments could become even more time consuming and cost prohibitive if thousands of chemically synthesized and modified oligonucleotides are required.
  • oligonucleotide therapeutics have become popular because they have the potential to therapeutically regulate essentially any gene of interest at the DNA or RNA level. Their versatility in treating inherited or acquired disorders stems from the ability to induce efficient gene silencing, gene expression or gene editing.
  • oligonucleotide as a therapeutic in vivo is challenging because of their unfavorable physicochemical characteristics.
  • oligonucleotides are susceptible to degradation by nucleases in the circulation, suffer from rapid renal clearance, and induce immunostimulatory effects via pattern recognition receptors, resulting in adverse effects.
  • oligonucleotide therapeutics that overcome the disadvantages outlined above.
  • the inventors have now disclosed techniques for selectively and effectively targeting and tagging the hydroxyl group at the 5’ and/or 3’ end of an oligonucleotide (for example, a DNA, or RNA) with a Phosphorus(V) (“P(V)”) based reagent, to thereby incorporate a small molecule handle linked via P(V) bond into the oligonucleotide.
  • P(V) Phosphorus(V)
  • the resulting modified oligonucleotide conjugate can be a stable, nuclease resistant biomimetic having a P(V) linkage.
  • the extraordinarivity and rapid reaction time of the methods and disclosed herein allows for site selective chemical modification of unprotected, native, DNA for the production of DNA based florescent probes, diagnostics, conjugates or therapeutics.
  • the oligonucleotide may be an unprotected or native. This method of oligonucleotide modification has been named SENDR by the inventors (Synthetic Elaboration of Native DNA by RASS).
  • a method for site selective chemical modification of an oligonucleotide comprising: immobilizing the oligonucleotide on a solid support to form oligonucleotide-solid support complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-solid support complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the solid support.
  • V phosphorus
  • the site-selective chemical modification of the oligonucleotide may comprise modification at 5’ hydroxyl group or 3’ hydroxyl group, or both 5’ and 3’ hydroxyl groups.
  • the phosphorus (V) reagent is contemplated to have a chemical structure
  • X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’, R’ is at each occurrence are independently selected from the group consisting hydrogen, C 1 to C 10 alkyl, C 1 to C 10 heteroalkyl, C 1 to C 10 cycloalkyl, C 1 to C 10 heterocycle, C 1 to C 10 aryl, C 1 to C 10 heteroaryl and C 1 to C 10 aralkyl
  • Y and Z are each independently selected from the group consisting of O, S, and N
  • R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO 2 , halogen, C 1 to C 20 alkyl, C 1 to C 20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted
  • the phosphorus (V) reagent has the chemical structure
  • the oligonucleotide may be a DNA, RNA, LNA, MPO, PMO, or derivations and chimeric mixtures thereof.
  • the modified oligonucleotide is preferably modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group.
  • the oligonucleotide may be structured aptamer, primer or hybridization probe, or a library thereof.
  • the oligonucleotide is further conjugated to a protein to form a oligonucleotide-protein conjugate.
  • the protein is preferably a therapeutic antibody.
  • the oligonucleotide is modified at multiple specific locations.
  • the method disclosed herein may further comprise eluting the modified oligonucleotide from the solid support by washing the oligonucleotide-solid support complex with an aqueous buffer solution.
  • the P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes.
  • the modified oligonucleotide may further conjugate with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins.
  • the biorthogonal chemistry may comprise Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide formation, amide coupling, proximity photoaffinity labeling, hydrazone ligation, and CuAAC reaction.
  • SPAAC Strain Promoted Azide Alkyne Cycloaddition
  • IEDDA Inverse Electron Demand Diels-Alder
  • unsymmetrical disulfide formation amide coupling
  • proximity photoaffinity labeling hydrazone ligation
  • CuAAC reaction CuAAC reaction.
  • the oligonucleotide comprises unnatural nucleotides.
  • the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
  • a method of increasing the half-life of an oligonucleotide therapeutic in-vivo comprising: immobilizing the oligonucleotide on an inert solid support to form oligonucleotide-solid support complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-solid support complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the inert solid support, wherein the site specific P(V) reagent modification increases stability and the half-life of the oligonucleotide therapeutic in vivo.
  • a method of targeting delivery of an oligonucleotide therapeutic to a specific location in the body of a patient comprising: attaching a sugar or lipid to a therapeutic oligonucleotide by the method of claim 1 to thereby produce a modified therapeutic oligonucleotide; and administering the modified therapeutic oligonucleotide to the patient for targeted delivery to a specific location in the patient body.
  • the site-selective chemical modification of the oligonucleotide comprises modification at 5’ hydroxyl group, or 3’ hydroxyl group or both 5’ and 3’ hydroxyl groups.
  • the phosphorus (V) reagent has a chemical structure , wherein X is a leaving group having the formula O-R’, S-R’, N-R’,
  • R’ is at each occurrence are independently selected from the group consisting hydrogen, C 1 to C 10 alkyl, C 1 to C 10 heteroalkyl, C 1 to C 10 cycloalkyl, C 1 to C 10 heterocycle, C 1 to C 10 aryl, C 1 to C 10 heteroaryl and C 1 to C 10 aralkyl
  • Y and Z are each independently selected from the group consisting of O, S, and N
  • R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO 2 , halogen, C 1 to C 20 alkyl, C 1 to C 20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxycarbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate
  • the oligonucleotide therapeutic is a DNA, RNA, LNA, MPO, PMO, or derivations or chimeric mixtures thereof.
  • the modified therapeutic oligonucleotide is modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group.
  • the therapeutic oligonucleotide is a structured aptamer or hybridization probe.
  • the therapeutic oligonucleotide is further conjugated to a protein to form an oligonucleotide-protein conjugate.
  • the protein is a therapeutic antibody.
  • the oligonucleotide is modified at multiple specific locations.
  • the method further comprises eluting the modified oligonucleotide from the solid support by washing the oligonucleotide-solid support complex with an aqueous buffer solution.
  • the P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes.
  • the modified oligonucleotide further conjugates with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins.
  • the biorthogonal chemistry comprises Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffinity labeling, hydrazone ligation, and CuAAC reaction.
  • SPAAC Strain Promoted Azide Alkyne Cycloaddition
  • IEDDA Inverse Electron Demand Diels-Alder
  • unsymmetrical disulfide Formation amide coupling
  • proximity photoaffinity labeling hydrazone ligation
  • CuAAC reaction CuAAC reaction.
  • the oligonucleotide comprises unnatural nucleotides.
  • the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
  • kits for DNA modification comprising:
  • the elution buffer comprises 1M NaC104, 20% MeOH, 40 mM Tris pH 8.5.
  • a method of diagnosing a disease in a patient in need thereof comprising: obtaining a tissue sample, blood sample, or body fluid swab from the patient; extracting oligonucleotides from the patient sample; labeling and/or modifying oligonucleotide with by the method of claim 1 that may be cognate to the disease of interest; performing PCR reaction on the labeled and/or modified oligonucleotide; and analyzing the labeled and/or modified oligonucleotide to diagnose the disease.
  • the disease is an infectious disease.
  • the disease is a bacterial or viral disease, and the patient is diagnosed with the disease if the analyzed labeled and/or modified oligonucleotide comprises the bacterial or viral DNA.
  • the disease is a tumor, and the patient is diagnosed with the tumor if the analyzed labeled and/or modified oligonucleotide comprises cancer specific DNA mutations.
  • the reactions disclosed herein may be used to create libraries or combinatorial libraries of ligated oligonucleotides linked through two chemical handles incorporated on the termini of the oligo.
  • this strategy could be sued to create circularized oligonucleotides.
  • this strategy could be used to assemble synthetic or natural genes or gene fragments
  • Fig. 1 depicts an exemplary modification of native DNA.
  • A prior art method of enzymatic stochastic labeling of DNA.
  • B prior art method of chemical stochastic labeling of DNA.
  • C Chemical modification of DNA, Phosphoramidate formation.
  • D chemical modification of oligonucleotide using the SENDR method disclosed herein.
  • Fig. 2 depicts exemplary ⁇ -modules synthesized for this study.
  • Fig. 3 depicts P(V) based DNA modification.
  • A SENDR enabled DNA modification.
  • B Optimization of the coupling step.
  • C Substrate scope. Conversions based on HPLC integration of a total absorbance signal at 260 nm. Unless otherwise noted, standard reaction conditions were applied; ⁇ -module (150 mM), DBU (450 mM), in dry MeCN (250 ⁇ L), 60 min, r.t. while adsorbed to Strata XL-A. a75 mM PSI and 225 mM DBU, b45 °C,c37 °C. d200 mM PSI at 50 °C. e300 mM PSI at 37 °C.
  • DNA loading (adsorption step) performed in PBS. Resin washed with DMA (x2) and THF (x3). Resin dried under a vacuum 2 h. Elution was performed using elution buffer 1 M NaC104, 40 mM Tris pH 8.5, 20% MeOH. In-situ protocol
  • FIG. 4 depicts exemplary downstream synthetic manipulations of SENDR-derived DNA-small molecule hybrids (ligated at 3' or 5')
  • FIG. 5 depicts exemplary SENDR compatibility with PS DNA and lager structured oligomers.
  • (Top) SENDR compatibility with PS DNA.
  • (Bottom) SENDR compatibility with larger structured oligomers.
  • Standard reaction conditions were applied; ⁇ -module (150 mM), DBU (450 mM), in dry MeCN (250 ⁇ L), 60 min, 37 °C while adsorbed to Strata XL-A.
  • Fig. 6 depicts exemplary SENDR aptamer modification.
  • A Standard reaction conditions were applied; ⁇ -module (150 mM), DBU (450 mM), in dry MeCN (250 ⁇ L), 60 min, r.t. while adsorbed to Strata XL-A.
  • B Direct incorporation of electrophiles into aptamers and their inhibition of protein targets. The reaction conditions that were applied: ⁇ -module (150 mM), DBU (450 mM), in dry MeCN (250 ⁇ L), 60 min, 37 °C, while adsorbed to Strata XL-A.
  • Fig. 7 depicts exemplary SENDR on biosynthetically derived DNA.
  • Scheme representing the biosynthetic steps to produce the COVID-19 N gene amplicon (59).
  • HPLC chromatogram of the SENDR reaction Deconvoluted mass spectrum of the starting material peak and the product peak.
  • Fig. 8 depicts exemplary SENDR enabled DNA-protein conjugation.
  • A DNA-BSA conjugation (ESI-TOF mass spectra of starting material and product).
  • B cTegsedi-DVD conjugation (SDS PAGE and catalytic methodol fluorescence assay including control experiments).
  • Fig. 9 depicts exemplary creation of dual labeled DNA probes.
  • A The synthesis of a TaqMan probe for RNaseP.
  • B Synthesis of the COVID 19 qPCR panel of probes.
  • Fig. 10 illustrates the LCMS Characterization of 61
  • Fig. 11 illustrates an embodiment of analysis of DNA-antibody (h38C2 IgG1) conjugation by the methods disclosed herein.
  • Fig. 12 illustrates the LCMS Characterization of SI-40
  • Fig. 13 illustrates the LCMS Characterization of 64.
  • Fig. 14 illustrates one embodiment of the methodol assay as used herein.
  • Fig. 15 illustrates SDS Page analysis of an antibody-DNA construct.
  • Fig. 16 illustrates the LCMS Characterization of compound 52.
  • the DNA corresponding to this compound is SEQ ID NO: 10
  • Fig. 17 illustrates the LCMS Characterization of compound 53.
  • the DNA corresponding to this compound is SEQ ID NO: 10.
  • the inventors have now discovered a method for non-covalent immobilization of an oligonucleotide, and then chemical modification the oligonucleotide at the native terminal hydroxyl groups.
  • This new site-specific oligonucleotide modification approach results in reliable and specific incorporation of a chemical moiety in the 5’ and/or 3’ end of a nucleic acid.
  • Reversible Adsorption of Solid Support is a process which allows for the adsorption of biomacromolecules onto a solid support to facilitate their transfer into solvents or reaction paradigms that would previously be considered incompatible.
  • DNA is adsorbed to a polystyrene based cationic support, through a simple mixing procedure, and the solvent exchanged (by simple washing and drying) into near anhydrous conditions to perform water incompatible reactions.
  • P(V) chemistry for the site- specific labeling of native oligonucleotide.
  • P(V) reagents include PSI, PS2, PO2, Rac-PSI, or PI.
  • a variety of P(V) ⁇ -loaded reagents ( ⁇ modules, Fig. 2) were prepared.
  • Model DNA 1 and 2 each contain a single modifiable terminal hydroxyl group and a single terminal phosphate which acts as a native protecting group ( Figure 3A).
  • Figure 3A the DNA sequences in Model DNA 1 and 2 are for illustrative purposes only, and any DNA or RNA sequences may be used in the methods disclosed herein.
  • one aspect of the present disclosure provides methods for site selective chemical modification of an oligonucleotide.
  • the method comprises immobilizing the oligonucleotide on an inert resin to form oligonucleotide-resin complex and contacting the oligonucleotide-resin complex with a phosphorus (V) based phosphorus-sulfur incorporation (PSI) reagent in an organic solvent to produce a modified oligonucleotide.
  • V phosphorus
  • PSI phosphorus-sulfur incorporation
  • the oligonucleotides are DNA or RNA, natural or unnatural.
  • solid support is the phase that is either a solid or liquid particle surface on which the components of the mixture to be separated is absorbed or adsorbed selectively.
  • the support remains stationary while the other phases move.
  • the solid support used herein comprise of porous materials, thus allowing the attachment of components during adsorption.
  • Many different types of solid support are contemplated in this present disclosure, such as gel beads, thin paper preferably thin uniform paper, silica, glass, gases, or liquid components. The exact nature of the solid support used to practice the methods of this disclosure would depend on the nature of the components to be separated.
  • nucleic acid “oligonucleotide” and/or “polynucleotide” are used interchangeably herewith and includes any nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)), analogs thereof, and polymers thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA.
  • RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides.
  • RNA poly- or oligo-ribonucleotides
  • DNA poly- or oligo-deoxyribonucleotides
  • RNA or DNA derived from N-gly cosides or C-gly cosides of nucleobases and/or modified nucleobases
  • nucleic acids derived from sugars and/or modified sugars and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as “intemucleotide linkages”).
  • the term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges.
  • nucleic acids containing ribose moieties examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties.
  • the nucleic acids, oligonucleotides, and polynucleotides as used herein may be naturally occurring, synthetically prepared, or biosynthetically prepared.
  • the terms “nucleic acid” “oligonucleotide” and/or “polynucleotide” includes Antisense oligonucleotides or oligomers (ASOs).
  • ASOs are short synthetic nucleic acid analogs that are being increasingly used as therapeutics. These oligonucleotides which bind RNA targets through Watson-Crick-Franklin base pairing, result in reduced gene expression or alterations in RNA processing, depending on their design and mode of action for the therapeutic.
  • PS Phosphorothioate
  • Second-generation modifications such as 2'-O- methyl (2'-O-Me), 2'-O-methoxy ethyl (MOE), constrained ethyl (cEt), and locked nucleic acid (LNA) further increase metabolic stability of oligonucleotides, as well as the binding affinity for RNA in vivo, conferring enhanced drug-like properties.
  • the present disclosure provides methods and techniques for making such modified oligonucleotides for increased stability (half-life).
  • nucleic acid “oligonucleotide” and/or “polynucleotide” as used herein further contemplates Phosphordiamidate (PDA) morpholino oligomers (PMOs).
  • PDA Phosphordiamidate
  • PMOs replace the pentose sugar with a morpholine ring, and the phosphate with a neutral PDA linkage.
  • PMOs exhibit similar binding affinity to RNA as DNA, have significantly enhanced metabolic stability as well as low protein binding due to the uncharged backbone, and do not support RNase H activity. The low protein binding feature also makes high-dose PMOs safer in vivo, compared to other chemistries.
  • nucleic acid “oligonucleotide” and/or “polynucleotide” as used herein further contemplates Locked Nucleic Acid (LNA) oligomeric compounds (also referred herein as LNA oligomers or LNA oligonucleotides).
  • LNA oligomer comprises at least one LNA nucleoside, such as a nucleoside which comprises a covalent bridge (also referred to a radical) between the 2' and 4' position (a 2'-4' bridge).
  • LNA nucleosides are also referred to as “bicyclic nucleosides”.
  • the LNA oligomer is typically a single stranded oligonucleotide.
  • the LNA oligomer comprises or is a gapmer. In some embodiments the LNA oligomer comprises or is a mixmer. In some embodiments the LNA oligomer comprises or is a totalmer. In some embodiments, the nucleoside analogues present in the oligomer are all LNA, and the oligomer may, optionally further comprise RNA or DNA, such as DNA nucleosides (e.g. in a gapmer or mixmer).
  • nucleic acid “oligonucleotide” and/or “polynucleotide” as used herein also contemplates Methylphosphonate (MP) oligonucleotides (MPOs).
  • MPOs are metabolically stable analogs of conventional DNA or RNA and contains a methyl group in place of one of the non-bonding phosphoryl oxygens. MPOs are highly resistant to metabolic breakdown in biological systems. Unlike natural phosphodiester oligonucleotides, MPOs contain chiral linkages.
  • nucleic acid “oligonucleotide” and/or “polynucleotide”, as used herein, are polymers of nucleosides joined, generally, through phosphodiester linkages, although alternate linkages, such as phosphorothioate esters may also be used in oligonucleotides.
  • a nucleoside consists of a purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U), or derivative thereof) base bonded to a sugar.
  • the four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine.
  • a nucleotide is a phosphate ester of a nucleoside.
  • nucleosides and nucleobases are known in the art and each of them are explicitly contemplated herein.
  • the nucleoside comprises a naturally-occurring nucleobase, such as adenine, guanine, cytosine, uridine, thymine, 5 -methyl cytosine, etc.
  • the nucleoside comprises other natural nucleobases, as well as modified nucleobases, such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5 -halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine.
  • modified nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl
  • nucleosides and nucleobases described herein include, but are not limited to the following: 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6- methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1- methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2- methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine;
  • aminoalkylaminocarbonylethylenyl (aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
  • aminocarbonylethylenyl-4 (thio)pseudouracil 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; l-(aminoalkylamino-carbonylethylenyl)-2- (thio)-pseudouracil; l-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine triphosphate; 1- methyl-3-(3-amino-3-carboxypropyl)pseudo- uridine triphosphate; 1 -methyl-pseudo- uridine triphosphate; 1 -ethyl-pseudo- uridine tri
  • Nucleosides described herein can also include modified sugars.
  • 2'-Sugar modifications of the present disclosure include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O- alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula (O-alkyl)m, where m is 1 to about 10.
  • Preferred among these poly ethers are linear and cyclic polyethylene glycols (PEGs), and PEG-containing groups, such as crown ethers and those which are disclosed by Ouchi et al, Drug Design and Discovery 1992, 9, 93; Ravasio et al. , J. Org. Chem.
  • Additional 2'-sugar modifications of use in the present invention include 2'-SR and 2'- NR.2 groups, where each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl.
  • 2'-SR nucleosides are disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in its entirety.
  • the present invention also includes nucleic acids derivatized with selenium (Se).
  • Se-derivatized nucleic acids include, but are not limited to, nucleic acids where O-atom at the positions 2', 4', and/or 5' of the sugar have been replaced with Se.
  • Other examples include oxygen replacement with Se in nucleobases and non-bridging phosphates.
  • Such nucleic acids are described in, for example, Pallan et al, Nat. Protoc., 2(3):647-51 (2007), and Nat. Protoc., 2(3);640-646 (2007), hereby incorporated by reference in their entirety.
  • nucleic acids suitable for the present invention include boron containing nucleic acids, such as those described in Schinazi et al., Nucleosides and Nucelotides, 17(635-647 (1998); Biochem., 35(18):5741-5746 (1996); J. Org. Chem., 79(8):3465-3472 (2014), hereby incorporated by reference in their entirety.
  • the methods disclosed herein further comprises washing the oligonucleotide-resin complex with an organic solvent after the immobilizing step.
  • the method also comprising drying the oligonucleotide-resin complex after the immobilizing step.
  • the method may also comprise eluting the modified oligonucleotide from the resin by washing the oligonucleotide-resin complex with an aqueous buffer solution, once the modification of the DNA is complete.
  • DNA starting material without terminal phosphates
  • DNA lacking a terminal phosphate could be readily phosphorylated by T4 polynucleotide kinase (T4PNK) (30 min) and directly loaded onto the support for subsequent SENDR protocol.
  • T4PNK T4 polynucleotide kinase
  • Biochemically phosphorylated substrates were labeled at efficiencies comparable to chemically phosphorylated material. The conditions also proved to be sequence independent.
  • SENDR provided efficient conjugations to oligonucleotides regardless of the identity of the terminal nucleoside.
  • P(V)-modules derived from alcohol nucleophiles could be prepared as stable (and often crystalline) reagents. Combined with the present findings, subsequent coupling to DNA sequences would allow for a vast scope. Indeed, a large number of P(V)-modules (Fig. 2, ⁇ - 1- ⁇ -20), including multiple click chemistry handles, protected amines, an activated disulfide, an MRI probe, a fluorescent quencher, a ligand for radiomedicine, photoaffmity tag, a fluorophore, and nucleosides, were prepared.
  • DNA-linked azides and alkynes were competent in SPAAC and CuAAC respectively, and directly provided constructs that were useful without further purification.
  • DNA-linked azides could be easily transformed into the corresponding amines through the addition of a water-soluble phosphine (TCEP). This manipulation could be performed after SENDR as a one-pot procedure in the elution buffer, providing an exceedingly simple route to amine-modified DNA.
  • P(V) and PSI reagents disclosed herein can be readily derivatized with almost any alcohol allowing for almost unlimited scope in the production of P(V) modifications or handles.
  • a large number of PSI or P(V) derived tags which included multiple click chemistry handles, protected amines, an activated disulfide, MRI probe, fluorescent quencher, radio medicine ligand, photoaffinity tags, and nucleosides, were developed. All these reagents were found to produce singly labeled product in good to excellent yields at both the 3’ and 5’ hydroxyl group with good DNA recovery (50-80%) ( Figure 3). Although the reaction conditions identified were readily implementable, some of these couplings proceeded better at 37°C, probably due to differences in solubility.
  • PSI reagent molecule contemplated herein may comprise, but is not limited to, the following chemical formulas:
  • PSI or P(V) reagents contemplated herein have a general formula wherein X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’,
  • R’ is at each occurrence are independently selected from the group consisting hydrogen, C 1 to C 10 alkyl, C 1 to C 10 heteroalkyl, C 1 to C 10 cycloalkyl, C 1 to C 10 heterocycle, C 1 to C 10 aryl, C 1 to C 10 heteroaryl and C 1 to C 10 aralkyl
  • Y and Z are each independently selected from the group consisting of O, S, and N
  • R 1 , R 2 , R 3 , and R 4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO 2 , halogen, C 1 to C 20 alkyl, C 1 to C 20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein: any two of R 1 , R 2 , R 3 , and R 4 , either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle.
  • the leaving group X reacts with a nucleophile to produce the PSI or P(V) module, , which is then used for reacting with the nucleotide.
  • P(V) reagents and their corresponding P(V) modules are shown below:
  • DNA linked azides were competent in SPAAC and DNA-linked alkynes were competent in CuAAC directly providing constructs that were experimentally useful.
  • DNA linked azides could be easily transformed into the corresponding amines through the addition of a water-soluble phosphine (TCEP).
  • TCEP water-soluble phosphine
  • the SENDR technology has the advantage of being applicable to a variety of oligonucleotides.
  • the inventors found that SENDR was able to efficiently modify both phosophothioate antisense oligonucleotides and large structured aptamers ( Figure 4).
  • the 3’ phosphorylated version of Fomivirsen (Vitravene), which is an antisense antiviral drug used in the treatment of cytomegalovirus retinitis in immunocompromised patients, was efficiently modified with a number of reactive handles without modification of the basic protocol.
  • Fomivirsen Fomivirsen
  • the inventors contemplate creating pools of Antisense Oligonucleotides with handles for the attachment of various target engaging small molecules or peptides.
  • the inventors found that a large (58 nt) protein A aptamer which exhibits significant secondary could also be routinely modified in good conversion. This could be useful in the modification of entire SELEX pools with reactive warheads or target engaging moieties.
  • the modified oligonucleotide is modified at the 5’ hydroxyl group. In other embodiments, wherein the modified oligonucleotide is modified at the 3’ hydroxyl group. In still other embodiments, the oligonucleotide is modified at both the 3’ end and the 5’ end. In such cases, the 5’ modification may be the same as the 3’ modification, or the 5’ modification may distinct and separate from the 3’ modification.
  • the oligonucleotide is a structured aptamer, a gene fragment, a primer, a hybridization probe, an oligonucleotide therapeutic, a protein recognition sequence, or a library of the aforementioned.
  • the nucleotide may also be conjugated to a protein to form an oligonucleotide-protein conjugate.
  • the method may further comprise incorporation of unnatural nucleotides into the nucleic acid sequence.
  • unnatural nucleotides include polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides
  • the PSI linked small molecules comprises reactive handles, affinity tags, fluorophores, and FMRI probes, as described above.
  • the modified oligonucleotide may further conjugate with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins.
  • the biorthogonal chemistry may comprise Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffmity labeling, and hydrazone ligation.
  • the method may further comprise chemical ligation of two strands of nucleic acids by reacting a first modified strand of the nucleic acid with another modified strand of nucleic acid using a biorthogonal reaction such as the CuAAC reaction.
  • the SENDR methods disclosed herein may also be used for diagnosing a disease in a patient in need thereof.
  • the method comprises obtaining a tissue sample, blood sample, or body fluid swab from the patient; extracting oligonucleotides from the patient sample; labeling and/or modifying the oligonucleotide by the SENDR method disclosed herein; performing PCR reaction on the labeled and/or modified oligonucleotide; and analyzing the labeled and/or modified oligonucleotide to diagnose the disease.
  • the disease may an infectious disease.
  • the disease is a bacterial or viral disease
  • the patient is diagnosed with the disease if the analyzed labeled and/or modified oligonucleotide comprises the bacterial or viral DNA.
  • the disease may be a tumor, and the patient is diagnosed with the tumor if the analyzed labeled and/or modified oligonucleotide comprises cancer specific DNA mutations.
  • the method may further comprise treating the patient with a known drug.
  • RASS makes use of non-covalent adsorption of DNA onto a solid support to facilitate solvent exchange from aqueous buffers, in which DNA is most soluble, into polar and nonpolar organic solvents that are otherwise considered as DNA incompatible.
  • organic solvent refers to any solvent except aqueous solutions.
  • organic solvent refers to any solvent containing carbon compounds. Examples of organic solvents include, but are not limited to, aromatic compounds, chloroform, alcohols, phenols, esters, ethers, ketones, amines, and nitrated and halogenated hydrocarbons.
  • RASS Reversible Adsorption to Solid Support
  • the RASS technique has several advantages over currently available methods. For example, a small molecule tethered to a DNA hairpin (headpiece domain) can be adsorbed to the solid support, transferred to organic solvent and undergo a range of chemical transformations in an organic solvent selected specifically for the chemical reaction, rather than to accommodate the encoding DNA.
  • Another advantage of the approach is that excess reagents and solvent can be washed away leaving the small molecule-DNA conjugate adsorbed to the resin. This property allows for sequential reactions such as two-step transformations and deprotection reaction sequences to be performed in a facile manner. Following the desired chemical modifications, the small molecule- DNA conjugate can be released from the solid support for subsequent mixing and pooling steps.
  • RASS includes a method of facilitating a chemical reaction in an organic solvent, comprising: providing a polynucleotide encoded chemical library, wherein the polynucleotide encoded chemical library members are immobilized to a solid support by non- covalent adsorption.
  • the polynucleotide encoded chemical library member is immobilized by non-covalent adsorption of the polynucleotide moiety to the solid support.
  • the polynucleotide is a DNA
  • the polynucleotide encoded chemical library is a DNA encoded chemical library.
  • the solid support comprises an anion exchange resin.
  • the solid support comprises a cation exchange resin.
  • the solid support comprises SDB-L and/or C18 silica gel.
  • the solid support comprises a hydrophobic resin.
  • the hydrophobic resin comprises a reversed phase, hydrophobic interaction, strong cation, weak cation, and affinity chromatography.
  • the solid support comprises silica-based resin, crosslinked polystyrene, crosslinked glycan, crosslinked PEG, and combinations thereof.
  • This procedure identified a strong-anion exchange resin to which aqueous DNA can be adsorbed, transferred, and retained in a variety of organic solvents including: trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), 1,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), 1 ,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH).
  • TFE trifluoroethanol
  • HFIP hexafluoroisopropanol
  • 1,4-dioxane 1,4-dioxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisoxane
  • THFIP hexafluoroisopropanol
  • THFIP hexafluoroiso
  • the organic solvent comprises trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), 1,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), 1,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH).
  • the immobilized polynucleotide as disclosed herein can undergo selective chemical modification while adsorbed to the solid support and exposed to organic solvent.
  • the chemical modification comprises an amide coupling in methylene chloride.
  • the chemical modification comprises a Suzuki coupling in dimethylacetamide.
  • the chemical library members undergo single or multiple synthetic reaction steps while being immobilized to the solid support.
  • the synthetic reaction steps may be in the same or in different solvents.
  • the method further comprises washing away small molecules and/or reagents while leaving the polynucleotide encoded chemical library immobilized to the solid support.
  • the RASS method may be used in challenging reactions such as decarboxylative cross couplings and CH activations.
  • the chemical composition of the linker between the DNA and the encoded small molecule may also be used to enhance the performance of a specific reaction.
  • anion exchange resin is highly promising, the inventors have found the current method to be compatible with a wide range of resins (anion exchange and others).
  • distinct resins should be used to facilitate specific desired chemical transformations. The technology would allow adaptation of previously incompatible reactants and chemistries and facilitate access to previously inaccessible chemical and conformational diversity.
  • the RASS method further comprises a covalent tag that modulates binding to the solid support and removal from the solid support.
  • the method further comprises a linker between the polynucleotide and the chemical library member.
  • the linker modulates steric or conformational interactions between the site of reaction, polynucleotide, and the solid support.
  • the linker facilitates the desired chemical reaction.
  • the solid support is modified with ligands, catalysts etc. to affect the desired reaction.
  • RASS Reversible Adsorption to Solid Support
  • a mixed mode polystyrene strong anion exchange resin (for example, Phenomenex, Strata-XA) which contains a butyl quaternary ammonium moiety, was found to be an excellent platform for RASS on DNA (Figure 5B).
  • Such resins incorporate both hydrophobic interactions (polystyrene, butyl substituents) and electrostatic interactions (quaternary amine) and effectively anchor the DNA in a polyvalent manner.
  • PBS 100 mM DNA
  • the bound DNA could then be eluted into a high salt elution buffer, an approach that is widely applied in molecular biology.
  • the Strata-XA resin allows the DNA- resin complex to be transferred from aqueous solution into neat organic solvents. Following removal of the solvent from the resin, the DNA can be washed with aqueous buffer, eluted from the resin with salt, and isolated through ethanol precipitation. Failures encountered with numerous other resins that were tested can be attributed to a lack of strong initial DNA binding or lack of retention in organic solvents. This represents a robust platform for the controlled, reversible binding and elution of DNA fragments to facilitate manipulation in organic media in ways that are not possible using conventional aqueous techniques.
  • SENDR could also be adapted for the efficient modification of phosphorothioate antisense oligonucleotides (ASOs) (Fig. 5).
  • ASOs phosphorothioate antisense oligonucleotides
  • FIG. 5 The 3' phosphorylated version of Vitravene, an FDA-approved ASO for the treatment of cytomegalovirus retinitis (CMV), was efficiently ligated with a number of P(V)-modules with no change to the general protocol.
  • CMV cytomegalovirus retinitis
  • MALAT1 a published ASO with multiple modified sugars and bases, and containing PS linkages, could be readily modified with multiple P(V)-modules. This result exemplifies the opportunity for the late-stage modification of ASO pools with target- engaging small molecules or peptides.
  • electrophile (Sp-(57)) and (Rp-(57)) conferred a 10-fold increase in potency (Ic50 of 15 and 17 nM respectively) when compared to parent aptamer (63) (Ic50 of 140 nM) in a fluorescence- based hNE inhibition assay. Also small molecules containing the same electrophiles were inactive in this screen.
  • This example further demonstrates the sequence and structure independence of the SENDR platform. It could prove useful in the modification of entire SELEX pools with libraries of reactive warheads or target engaging moieties for the facile creation of DNA-small molecule chimeric inhibitors.
  • SENDR Another utility of SENDR was also demonstrated in the formation of oligonucleotide- protein conjugates, which are becoming increasingly valuable in the production of long-acting and/or targeted oligonucleotide drugs.
  • An oligonucleotide was modified by SENDR (using ⁇ - 8) with an activated disulfide group resulting in (61) (Fig. 8A).
  • This construct could be used directly, without purification in a disulfide forming reaction with bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the reaction cleanly furnished the DNA-BSA conjugate (62), and ESI-TOF analysis of the crude reaction mixture indicated that no unmodified BSA remained in solution.
  • SENDR was also used to create a DNA construct that could be used in site-specific antibody conjugations (Fig. 8B).
  • the complementary cDNA sequence of FDA-approved ASO Tegsedi (cTegsedi) was modified with an alkyne handle. In turn, this alkyne was ligated to a reactive beta-lactam containing moiety via CuAAC.
  • the beta lactam containing oligonucleotide (64) was competent in the site-specific labeling of an engineered lysine on the heavy chain of a IgG that has in use in antibody drug conjugates (ADCs).
  • ADCs antibody drug conjugates
  • This system was derived from the antihapten mAh h38C2 and is especially reactive toward beta lactam haptens.
  • the reactive lysine residue also catalyzes a retro aldol reaction with methodol, which results in increased fluorescence of the aldehyde product.
  • the modified site on the antibody was confirmed to be the catalytic lysine via methodol florescence assay — after conjugation, signal from the florescent aldehyde was not detected (Fig. 8B).
  • the SENDR technology as disclosed herein provides access to oligonucleotide-protein conjugates.
  • SENDR enables formation of oligonucleotide protein conjugates, which are becoming increasingly valuable in the production of long acting and or targeted oligonucleotide drugs.
  • BSA bovine serum albumin
  • SENDR was also used to create a DNA construct that could be readily used in site specific antibody conjugations.
  • the complementary DNA sequence of approved ASO Tegsedi (cTegsedi) was modified with an alkyne handle to which a reactive beta-lactam containing moiety was clicked on.
  • This beta lactam containing DNA was competent in the site specific labeling of an engineered lysine on the heavy chain of an IgGthat is especially reactive toward beta lactam heptans. This lysine can also catalyze a retro aldol reaction and will cause in increase in fluoresces of a fluorogenic aldehyde derived from methadol.
  • the modified site on the antibody was confirmed to be the catalytic lysine by a methadol florescence assay in which signal from the florescent aldehyde was not detected.
  • These antibodies have shown promise as flexible platforms for the production of antibody drug conjugates, as they can be produced by typical recombinant methods and drug molecules can be added at a known stoichiometry, to a known position. Labeling the antibody with cTegsedi created, in effect, an ASO delivery system that would protect, target and deliver Tegsedi to the cell of interest. This process would be useful in the creation of many antibody-ASO conjugates that could provide targeted ASO therapy.
  • oligonucleotide-based technologies only require a single probe, the true power of hybridization probes is realized in dual labeled form.
  • the canonical dual labeled oligonucleotide probe has a fluorophore label on one terminus and a fluorescence quencher at the other (Fig. 9A). Fluorescence of the probe is quenched when the two components are in close proximity.
  • Molecular beacons for example, form a stem loop system that brings the termini labels into close proximity when the target is not present (Fig. 1A). Upon target engagement, the MB adopts an extended conformation, which moves the two labels out of
  • probes are also typically constructed with a fluorophore and a quencher at the termini. These probes hybridize to a diagnostic sequence of interest and upon PCR elongation by Taq polymerase, the probe is cleaved (by the intrinsic exonuclease activity of Taq), and increased fluorescence is read out (Fig. 1A).
  • TaqMan PCR is widely considered to be the state-of-the-art in real-time PCR methods, practitioners are reliant on vendors for custom synthesis of probes with proprietary linking technologies. This synthesis must be done for each individual target and can prove to be prohibitively expensive. Indeed, the less sensitive method of SYBR Green-based qPCR, which relies on increased fluorescence of an intercalating dye during polymerization, is gaining in popularity because of the immense cost of buying custom TaqMan PCR probes for every experiment. SENDR, when used in concert with ubiquitous biochemical techniques, presents a unique opportunity for biochemical researchers to produce dual labeled probes for their own custom applications (Fig. 9A). Synthesis of these probes proceeds through a multistage process.
  • a typical synthetic oligonucleotide containing a 5' phosphate and a 3' hydroxyl group is ligated with an alkyne group by SENDR (Fig. 9).
  • this modified oligomer is quantitatively dephosphorylated by recombinant shrimp alkaline phosphatase (rSAP) unmasking the 5' hydroxyl group, while the unmodified DNA is selectively degraded by ExoIII in one pot.
  • This oligomer is then subjected to a second SENDR modification at the 5' terminus, providing the dual labeled probe.
  • oligonucleotide is selectively degraded by a mixture of lambda exonuclease and T4PNKin one pot.
  • the T4PNK phosphorylates the remaining unlabeled 5' hydroxyl oligonucleotide which is then recognized and degraded by lambda exonuclease.
  • These resulting probes are highly pure without the need for HPLC purification.
  • the position of each label on the dual labeled probe was confirmed by MS fragmentation.
  • This dual-labeled parent probe (68) is now primed for subsequent SPAAC/CuAAC reactions with any fluorophore/quencher pair desired to furnish the qPCR- competent probe.
  • the SENDR technology was found to be useful for synthesis of dual labeled probes.
  • many oligonucleotide based technologies only require a single probe, the true power of a oligonucleotide probe is realized in the dual labeled form.
  • the canonical dual labeled probe has a fluorophore label on one terminus and a fluorescence quencher at the other. Fluorescence of the probe is quenched when the two components are in proximity.
  • Molecular bacons for example, form a stem loop system which brings the termini labels are in proximity when the target is not present.
  • the MB adopts an extended confirmation taking the two labels out of FRET range and resulting in a fluorescence signal.
  • TaqMan PCR probes Another ubiquitous example of these dual labeled probes is the TaqMan PCR probes. These probes are also typically constructed a fluorophore and a quencher at the termini. These probes hybridize to the diagnostic sequence of interest and upon PCR elongation by Taq polymerase the probe is cleaved (by the intrinsic exonuclease activity of Taq) and increased fluorescence is read out.
  • TaqMan PCR is widely used, the requisite dual labeled probes have to be custom synthesized for each target of interest, which in turn can prove to be expensive.
  • the SENDR method may be used for diagnosing a disease.
  • the disease may be an infectious disease, such as a bacterial infection or viral infection.
  • the SENDR protocol for diagnosing a viral disease, a coronavirus infection such as COVID-19 is described herein.
  • SENDR method was used to make dual ladled probes for COVID-19 diagnostics.
  • SENDR could provide health professionals and researchers with a time efficient probe source allowing continued therapeutic and vaccine research.
  • an exemplary compounds useful as a probe for coronavirus qPCR comprise the following, wherein N3 is an oligonucleotide from a coronavirus. The corresponding sequence listing is SEQ ID NO: 1.
  • a swab is taken from the nostril or throat of the patient. RNA present in the swab is extracted, and analyzed using the labeled PCR probe as disclosed throughout the disclosure. A PCR reaction is then performed to determine if coronavirus RNA is present in the patient sample.
  • the coronavirus disease is COVID-19.
  • the inventors contemplate the use of SENDR reagents in a kit format.
  • the process is simple and robust to be miniaturized and performed in a cartridge/flow set up and all reagents employed are shelf stable indefinitely.
  • the inventors found that reaction set up when performed in a cartridge proved simpler and faster than the typically employed microcentrifuge tubes. Also, reaction efficiency was identical to reactions performed in microcentrifuge tubes.
  • a kit is provided for oligonucleotide modification.
  • the kit comprises one or more P(V) reagents as discussed throughout this disclosure; 1,8-Diazabicyclo[5.4. 0]undec-7-ene (DBU); Dry Tetrahydrofuran (THF); Dimethylacetamide (DMA); Elution buffer; and Strata AXL resin in fitted cartridge.
  • the elution buffer comprises 1M NaCIO 4 , 20% MeOH, 40 mM Tris pH 8.5.
  • SENDR kit From a pragmatic standpoint, a simple kit-format would be of use to the community. Toward that end, a “SENDR kit” was created from readily available consumables. The SENDR process is simple and robust enough to be miniaturized and performed in a cartridge/flow set up, and all reagents employed are shelf-stable indefinitely. Gratifyingly, when performed in a cartridge, the process proved simpler and faster than the previously employed microcentrifuge tubes. Importantly, reaction efficiency was identical to reactions performed in microcentrifuge tubes. Using this kit, a researcher could customize synthetic or biochemically derived oligonucleotide, in-house, with a suite of commercialized reagents. We believe that SENDR kits will expand the toolbox and allow researchers to pursue experimental designs that were previously out of reach.
  • SENDR has the ability to easily make site-specific oligonucleotide modifications.
  • SENDR it does not require de novo synthesis (and specialized equipment) and benefits from the intrinsic advantages of a late-stage incorporation.
  • enzymatic means of site-selective functionalization are powerful, SENDR is uniquely versatile and programmable using easily accessible reagents. As more modules are developed, one could imagine an unlimited diversity being incorporated.
  • highly lipophilic groups are challenging to employ, groups sensitive to DBU might be problematic, and at this point are limited to terminal modifications.
  • the modular nature of the process could permit a more medicinal chemistry mindset into the derivatization of complex oligonucleotide-based conjugates.
  • SENDR-based modification of oligonucleotide lies outside the skill set of the general molecular biology practitioner, the robust chemistry should prove simple enough for any practitioner with basic liquid handling skills.
  • the present disclosure demonstrates conjugations that are compatible with simple organic molecules, proteins, aptamers, and ASOs. Numerous extensions such as applications to carbohydrate conjugation, and multiplexed high throughput array is possible and contemplated herein.
  • nucleic acid therapeutics In contrast to small molecule drugs and biologies which target gene products (i.e. proteins), nucleic acid therapeutics have the potential to therapeutically regulate essentially any gene of interest at the DNA or RNA level. Their versatility in treating inherited or acquired disorders stems from the ability to induce efficient gene silencing (inhibiting pathological/mutant protein production), gene expression (producing therapeutic proteins) or gene editing (correcting dysfunctional/mutated genes).
  • Nucleic acid therapeutics or oligonucleotide therapeutics include antisense oligonucleotides (ASO), small interfering RNA (siRNA), plasmid DNA (pDNA), messenger RNA (mRNA), and complexes containing guide RNA (gRNA) as part of gene editing approaches.
  • ASO antisense oligonucleotides
  • siRNA small interfering RNA
  • pDNA plasmid DNA
  • mRNA messenger RNA
  • gRNA complexes containing guide RNA
  • oligonucleotide as a therapeutic in vivo is challenging because of their unfavorable physicochemical characteristics, such as negative charge and relatively large size, which prevents their efficient uptake into cells.
  • oligonucleotides are susceptible to degradation by nucleases in the circulation, suffer from rapid renal clearance, and induce immunostimulatory effects via pattern recognition receptors, resulting in adverse effects.
  • these oligonucleotide therapeutics are potentially immunogenic and may require a delivery vehicle for efficient and specific transport to target cells and across the lipid bilayer.
  • the SENDR method of oligonucleotide modification as presented herein is able to overcome a number of these disadvantages inherently present in the currently known oligonucleotide therapeutics.
  • Lipids and sugars are commonly used for targeted delivery of oligonucleotide therapeutic to a specific location in the patient body.
  • lipid nano particle LNP
  • the modification at the 2'-position of the furanose sugar have been especially useful for improving the drug-like properties of antisense oligonucleotides (ASOs).
  • ASOs antisense oligonucleotides
  • the SENDR method as disclosed herein may be used to couple a sugar or lipid molecule to the oligonucleotide therapeutic to thereby target the delivery of the therapeutic oligonucleotide to the specific diseased cell location in the patient body.
  • the instant disclosure provides a method of targeting delivery of an oligonucleotide therapeutic to a specific location in the patient body, comprising: attaching a sugar or lipid to a therapeutic oligonucleotide by the SENDR method to thereby produce a modified therapeutic oligonucleotide; and administering the modified therapeutic oligonucleotide to the patient for targeted delivery to a specific location in the patient body.
  • DNA and RNA-based therapeutics are also inherently unstable and prone to degradation by active and abundant nucleases (DNases and RNases). This hurdle of a short half life may be overcome by chemically modifying DNA or RNA to enhance its stability, and by employing synthetic carriers such as lipid nanoparticle (LNP) or polymer-based nanoparticle (PNP) systems for drug delivery.
  • LNP lipid nanoparticle
  • PNP polymer-based nanoparticle
  • the present disclosure provides a method of increasing the half-life of an oligonucleotide therapeutic in-vivo, the method comprising: immobilizing the oligonucleotide on an inert resin to form oligonucleotide-resin complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-resin complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the inert resin, wherein the site specific P(V) reagent modification increases the half life of the oligonucleotide therapeutic in vivo.
  • modified oligonucleotides prepared by the method above is contemplated to have stabilizing moieties such as modified nucleosides including pseudouridine, 5’-methyl- cytidine triphosphate (m5CTP), N6-methyl-adenosine-5’- triphosphate (m6ATP), 2-thio- uridine triphosphate (s2UTP), N6-methyladenosine (m6A), and N6,2-Odimethyladenosine (m6Am), a 5 ’cap, optimized 3’ poly (A) tail etc.
  • stabilizing moieties such as modified nucleosides including pseudouridine, 5’-methyl- cytidine triphosphate (m5CTP), N6-methyl-adenosine-5’- triphosphate (m6ATP), 2-thio- uridine triphosphate (s2UTP), N6-methyladenosine (m6A), and N6,2-Odimethyladenosine (m6Am), a 5
  • the modified oligonucleotides as prepared by the SENDR method would enhance the stability of the oligonucleotide drug, provide protection from nuclease degradation, confer drug-like properties to DNA and RNA, reduce immune stimulation, maximize on-target potency and prolong the duration of the drug.
  • Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), and dichloromethane (DCM) and acetonitrile (MeCN) were obtained by passing the previously degassed solvents through an activated alumina column.
  • DMA was purchased from Sigma-Aldrich and used without further drying.
  • P(V) reagents were purchased from Sigma Aldrich or synthesized as described below.
  • DIC N,N’-diisopropylcarbodimide
  • Deionized water was used in all the reactions, unless otherwise stated. All the other reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated.
  • NaC104 was purchased at the highest commercial grade from Acros Organics. DNA tags was obtained from IDT, Inc., Coralville, IA. Recombinant All enzymes (Shrimp Alkaline Phosphatase (rSAP), ExoIII, T4PNK, and Lambda Exo) was obtained from New England Biolabs, Ipswich, MA. The Cut Smart buffer stock used enzymatic reactions was obtained from New England Biolabs, Ipswich, MA. UltraPureTM Agarose was obtained from Invitrogen, Carlsbad, CA. 50X TAE Buffer (Tris-acetate-EDTA) was obtained from Thermo Fisher Scientific, Waltham, MA. SYBRTM Safe DNA Gel Stain (10,000X) was obtained from Invitrogen, Carlsbad, CA. Gel Loading Dye, Purple (6X), no SDS was obtained from New England Biolabs, Ipswich, MA.
  • rSAP Shrimp Alkaline Phosphatase
  • Yields under normal organic conditions refer to chromatographically and spectroscopically (1H, 31P NMR) homogeneous material, unless otherwise stated.
  • TLC was performed using 0.25 mm E. Merck silica plates (60F-254), using short-wave UV light as the visualizing agent, and phosphomolybdic acid or KMn04 and heat as developing agents.
  • NMR spectra were recorded on Bruker DRX-600, DRX-500, and AMX-400 instruments and are calibrated using residual undeuterated solvent (CHC13 at 7.26 ppm 1H NMR, 77.16 ppm 13C NMR).
  • PBS Phosphate buffered saline was purchased from commercial and filtered before use. PBS is comprised of NaCl (137 mM), KC1 (2.7 mM), Na2HP04 (10 mM), KH2P04 (1.8 mM), at pH 7.4.
  • Elute Buffer DNA was eluted from resin using a sodium perchlorate buffer (1 M NaC104, 40 mM Tris, 20% MeOH, at pH 8.5). This was prepared from NaC104 (Acros Organics), and filtered after preparation. This buffer could be stored on the bench indefinitely.
  • Preparative HPLC was performed on the Waters H Class instrument described above using customized gradients for each compound of interest and an automatic divert valve.
  • the ⁇ -modules were prepared as follows: Alcohol (1.0 equiv.) and (-)- ⁇ (1.3 equiv.) were dissolved in anhydrous MeCN or DCM (0.1 M) in a flame-dried round-bottom flask. DBU (1.2 equiv.) was added to the reaction mixture while stirring. After 5-10 minutes, the crude reaction mixture was diluted with EtOAc or Et20 and transferred to a separatory funnel. The organic layer was washed with H20, saturated aqueous KH2P04 and brine. After drying over MgS04 and filtration, the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography unless otherwise stated
  • a module in solution it may be advantageous to directly prepare a module in solution and immediately use it in the SENDR process without isolation.
  • Strata-XAL if designed for large analytes.
  • Strata-XAL is a 100 pm particle a with 300 A pore size.
  • “Washing the resin” refers to adding 500 to 1000 ⁇ L of solvent to the resin, vortexing (30 sec), spinning the resin down, and removing the supernatant with a pipette. Dry acetonitrile should be used for best reaction conversion.
  • the elute buffer which contains 1 M NaClO 4 , 20% MeOH, 40 mM tris, pH 8.5 should be accessed with high quality NaClO.
  • flow throughs should be allowed to occur under gravity not under vacuum.
  • Agitating the resin can be done with an end over end tumbler or an orbital shaker. Drying the resin under vacuum can be performed using a lyophilizer, high vac, or speed vac (if the columns fit).
  • N refers to any unspecified nucleotide, and may be guanine or adenine or thymine or cytosine or uracil, or any modifications thereof:
  • SENDR was optimized by performing reactions in a microcentrifuge tube as described in General Procedure 2 with various reaction conditions (as described in the main text and Figure 2B). Briefly, 5 nmol of DNA was loaded onto 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum. In the case in which stringent drying was not performed, the loaded resin was simply washed with acetonitrile (500 ⁇ L three times).
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (-1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the supernatant ethanol was decanted off. The tubes were dried via speed vacuum, the DNA was dissolved, and HPLC-MS analysis was performed.
  • SENDR was also performed on High Tm Hairpin.
  • a hairpin oligo was incubated for 10 minutes at either RT or 66°C in 100 ⁇ L PBS. This solution was pipetted directly onto washed support (50 ⁇ L) and quickly vortexed. The reactions were allowed to cool to room temperature before washing and drying was performed according to general procedure 2.
  • SENDR derivatization was performed under standard conditions.
  • the SENDR method as disclosed herein may be used for various alcohol selective reactions.
  • the following alcohol selective reactions are described; however it should be recognized that the present disclosure is not limited to the reagents or reactions below, and these are merely presented as examples.
  • DNA 2 was dissolved (90 mM) in MES buffer (50 mM) at pH 6.2 containing phenethylaime (5 mM) and imidazole (20 mM). to this solution was added l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) (10 ⁇ L) from a stock solution in DMA (50 mM). This reaction was quickly vortexed and allowed to incubate at room temperature for 60 minutes. After 60 minutes the DNA was isolated by ethanol precipitation (previously described) and analyzed by liquid chromatography-mass spectrometry (LCMS).
  • MES buffer 50 mM
  • imidazole 20 mM
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. Phorphoramitide (150 mM) and tetrazole (450 mM) were added in dry MeCN and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resi was washed once with dry MeCN (500 ⁇ L) and oxidation solution was added (iodine, pyridine, water in THF). This was reaction incubated for 15 minutes and then the resin bed washed with MeCN and PBS. The DNA was eluted and precipitated as previously described and finally analyzed by LCMS.
  • DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. Tosyl Chloride (150 mM) and Collinide (450 mM) were added in dry MeCN and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resin was washed once with dry MeCN (500 ⁇ L). The DNA was eluted and precipitated as previously described and finally analyzed by LCMS.
  • DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. Nirtopheol (150 mM) and Triphenylphosphine (150 mM) were added in dry MeCN. DIAD (150 mM) was then spiked in and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resin was washed once with dry MeCN (500 ⁇ L). The DNA was eluted and precipitated as previously described and finally analyzed by LCMS. Low DNA recovery was observed by UV Vis absorbance (nanodrop). [00137] DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly.
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the supernatant ethanol was decanted off. The tubes were dried via speed vacuum, the DNA was dissolved and HPLC-MS analysis was performed.
  • Nonphosphorylated DNA was phosphorylated at the 5’ end by T4PNK. Briefly, 5 nMol DNA was added to a PCR tube and contiaining CutSmart (IX), 1 mM ATP, and 5 mM DTT (from 5x stocks). T4PNK was added (2 uL) and the reaction volume was brought to 50 ⁇ L total. The reaction tube was allowed to incubate for 30 minutes at 37°C. After 30 minutes the reaction mixture was added directly to the Strata XL-A support in PBS (500 uL) and vortexed. The support was dried according to General Procedure 2, and standard SENDR conditions were applied.
  • SENDR was optimized by performing reactions in microcentrifuge tube as described in General Procedure 2. Briefly, 5 nmol of DNA was loaded onto 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum.
  • DBU 450 mM, 18 ⁇ L
  • the reaction tube was vortexed for 30 seconds and incubated at room temperature for various amounts of time.
  • the reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 ⁇ L) and 1:1 MeCN:PBS (500 ⁇ L).
  • Elute buffer 300 ⁇ L was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes.
  • the DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolated via ethanol precipitation.
  • Ethanol precipitation was performed by adding 10% v/v of aNaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed. The method above was performed on a variety of substrates and a variety of DNA. Some of the modified oligonucleotides obtained by this method are illustrated below in table 1, and the corresponding sequence listings are SEQ ID NOs:2-5. Table 1.
  • Protocol for SENDR on PS DNA SENDR phsophorothioate (PS) compatibility was analyzed by performing reactions in microcentrifuge tube as described in General Procedure 2. Briefly, 5 nmol of PS DNA 46 was loaded onto 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum.
  • PS SENDR phsophorothioate
  • Ethanol precipitation was performed by adding 10% v/v of aNaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum, the DNA was dissolved, and HPLC-MS analysis was performed.
  • Exemplary modified oligonucleotides produced by this method comprise
  • Aptamer compatibility protocol SENDR Aptamer compatibility was analyzed by performing reactions in microcentrifuge tube as described in General Procedure 2. Briefly, 5 nmol of aptamer 54 was loaded onto 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum.
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed.
  • Exemplary modified oligonucleotides produced by this method comprise the following structure and SEQ ID NOs:6-7.
  • Protocol R/S-(57) were used directly in the assay after isolation, without further purification.
  • hNE human neutrophil elastase
  • Residual proteolytic activity was measured kinetically at 25 °C using a Synergy HI microplate reader (BioTek) for a total of 30 min at 30 sec intervals (Excitation: 380 nm, Emission: 460 nm). Only data points reflecting linear substrate conversion were used to determine relative protease activity. IC 50 values were obtained by fitting the data to a concentration-response inhibition, log (inhibitor) vs. response -variable slope (four parameters) using GraphPad Prism.
  • a biosynthetically derived single stranded DNA was obtained. PCR amplification was performed on this biosynthetically derived DNA.
  • PCR Amplification To a PCR tube template strand (2 ⁇ L, 5 ng from 2 ng/ ⁇ L stock), primer 1 (2.5 ⁇ L from a 10 ⁇ M stock), primer 2 (SI-20) (2.5 ⁇ L from a 10 ⁇ M stock), water (18 ⁇ L) were added on ice. To this mixture Q5 High Fidelity 2x Master Mix (NEB) was added (25 ⁇ L). Tubes were place into a preheated (95°C) thermocycler. An initial denaturing step of 95 °C for 30 was performed.
  • NEB High Fidelity 2x Master Mix
  • Lambda Exonuclease Degradation To the DNA mixture from the previous step (40 ⁇ L), CutSmart (5 ⁇ L, lOx), water (3 ⁇ L), and Lambda Exonuclease (2 ⁇ L) was added in a PCR tube. This mixture was vortexed and incubated at 37°C for 30 minutes at which point the lambda exonuclease was thermally denatured at 75°C for 10 minutes. The resulting ssDNA (not phosphorylated) was isolated using a ZymoSpin Oligo clean and concentrator column.
  • T4PNK Phosphorylation To the DNA mixture from the previous step (30 ⁇ L), CutSmart (5 ⁇ L, 10x), DTT (5 ⁇ L, 50 mM Stock), ATP (5 ⁇ L from 10 mM stock), water (3 uL) and T4PNK (2 ⁇ L) was added in a PCR tube. This mixture was vortexed and incubated at 37°C for 60 minutes at which point the T4PNK was thermally denatured at 75°C for 10 minutes. The resulting ssDNA was isolated using a ZymoSpin Oligo clean and concentrator column.
  • DNA was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 60 was loaded onto two tubes each containing 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum. These tubes were manipulated in parallel.
  • DBU 450 mM, 18 ⁇ L
  • the reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes.
  • the reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 ⁇ L) and 1:1 MeCN:PBS (500 ⁇ L).
  • Elute buffer 300 ⁇ L was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes.
  • the DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation.
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (- 20°C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC- MS analysis was performed.
  • DNA SI-40 (SEQ ID NO: 8) was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 63 was loaded onto two tubes containing 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum. These tubes were manipulated in parallel. [00163] ⁇ -5 in MeCN (150 mM, 250 ⁇ L) was added to the loaded and dried resin. Then
  • DBU 450 mM, 18 ⁇ L
  • the reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes.
  • the reaction was worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 ⁇ L) and 1:1 MeCN:PBS (500 ⁇ L).
  • Elute buffer 300 ⁇ L was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes.
  • the DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation.
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed.
  • DNA-Antibody (h38C2 IgGl) conjugation procedure Antibody in storage solution was buffer exchanged into PBS by amicon spin filter (30 kDA) and concentrated to 25 mM ( ⁇ 5 mg/mL). This solution was used to dissolve (50 ⁇ L) solid DNA to a presumptive concertation of 300 mM. In addition, a second tube containing 50 ⁇ L of Antibody was aliquoted and taken through an identical process to serve as a positive control. Dissolving 64 with antibody solution was performed by vortexing the solution. This reaction mixture was incubated at 37 °C for 8 hours to produce DNA-Antibody conjugate. The crude reactions mixture was analyzed for conjugation efficiency as previously described by Rader et. al.
  • Methodol assay The methodol assay for conjugation confirmation was performed as described by Rader and co-workers. Briefly, aliquots (12.5 ⁇ L) of the reaction and control solutions were diluted (0.2 mg/mL relative to original antibody concentration) in PBS to a final volume of 310 ⁇ L. Each sample were dispensed (98 ⁇ L) in triplicate into a black 96-well plate. Three blank wells, containing PBS were also dispensed (98 ⁇ L) into the black plate. A plate reader was prepared, the wavelength of excitation ( ⁇ ext) was set to 330 nm and wavelength of emission (Lem) was set to 452 nm.
  • the instrument was programed to record every minute for 60 minutes and shake the plate in between. Finally, methodol (10 mM in ethanol) was added (2 ⁇ L) to each well using a multichannel pipette and the plate was immediately loaded into the plate reader and data collection initiated. Signal was determined by normalizing against the blank wells. Measurements in triplicate were averaged and plotted along with standard deviation. Standard deviation was usually smaller than the marker size.
  • SDS PAGE Analysis SDS Page was performed to confirm mass increase of the antibody-DNA construct. For this analysis 1 pg of the reaction and the control were aliquoted into PCR tubes. To these tubes 6X Lamelli buffer (6 ⁇ L) was added and finally the tubes were diluted with water (to 24 ⁇ L). These reactions were heated at 95 °C for 10 minutes before being loaded into separate lanes on a precast Bio-Rad 4-20% SDS PAGE Gel. To another lane of
  • Bio-Rad Precsion Plus Protein standard was added (7 ⁇ L). The rest of the lanes were loaded with 6x Lamelli buffer (6 ⁇ L). The gel was run at 200V for 30 minutes at which point it was stained with Coomassie protein stain and distained with water over night. Finally, the gel was imaged on a Bio-Rad gel imager.
  • SENDR was used to access the dual label probes.
  • the first tag was appended to the 3’ end of DNA using General Procedure 2. Briefly, 25 nmol of precursor was loaded onto 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum. ⁇ -5 in MeCN (150 mM, 250 ⁇ L) was added to the loaded and dried resin. Then DBU (450 mM, 18 ⁇ L) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at 37 °C for various amounts of time.
  • the reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 ⁇ L) and 1:1 MeCN:PBS (500 ⁇ L). Elute buffer (300 ⁇ L) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20°C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed.
  • the intermediate product DNA was loaded onto resin and modified with SENDR by General Procedure 2 using ⁇ -17 to generate the dual labeled DNA product. After this reaction, the crude reaction mixture is ethanol precipitated, pelleted, dried, dissolved and analyzed by HPLC-MS.
  • Lambda Exo Cleanup The dual labeled DNA probe was loaded into a PCR tube (5 ⁇ g) in 30 uL water. To each tube CutSmart (5 ⁇ L at 10x), ATP (5 ⁇ L at 5 mM) and DTT (5 ⁇ L at 50 mM) was added. Finally Lambda exonuclease (2.5 ⁇ L) and T4PNK (2.5 ⁇ L) were added. These tubes were incubated for 90 minutes at 37°C at which point the enzymes were thermally deactivated at 75°C for 10 minutes.
  • the dual labeled DNA was added to a PCR tube in 38 uL water.
  • DMSO 8 uL
  • Tris buffer (2 ⁇ L, 1M at pH 8.5) and DBCO Fluorescein (2 ⁇ L, 10 mM, in DMSO) were added. This reaction was allowed to incubate at 37°C for 120 minutes and excess regents were removed by zymo spin column. Resulting in FAM labeled probe.
  • SEQ ID NO:9 An exemplary dual labeled DNA (SEQ ID NO:9) prepared by the above method is shown below:
  • DNA 3 was synthesized in cartridges according to General Procedure 3. Briefly, the resin (100 ⁇ L) was loaded into a fritted cartridge (Bio-Rad, Bio-Spin column, 1.2 mL, Cat. Num. 7326008). PBS (500 ⁇ L) was added to the resin bed and allowed to flow through with gravity. The column was capped on the bottom and DNA was loaded (5 nmol in 200 ⁇ L PBS) onto the resin bed. The column was then capped (on top), vortexed and agitated for 5 minutes. The load buffer was allowed to flow out with gravity.
  • PBS 500 ⁇ L
  • the column was capped on the bottom and DNA was loaded (5 nmol in 200 ⁇ L PBS) onto the resin bed. The column was then capped (on top), vortexed and agitated for 5 minutes. The load buffer was allowed to flow out with gravity.
  • the Resin bed was washed with DMA (500 ⁇ L twice) and THF (500 ⁇ L three times), and the solvent was allowed to flow through with gravity each time.
  • the top cap was replaced, and the resin was dried for 2 hours under vacuum (placed on a lyophilizer).
  • a new cap was replaced on the bottom and ⁇ -l was added (300 mM in MeCN, 125 ⁇ L) and then DBU was added (900 mM in MeCN, 125 ⁇ L).
  • the top cap was replaced, the cartridge was vortexed and the column was incubated at 37 °C for 60 minutes. After the reaction the caps were removed, and the reaction mixture was allowed to flow to waste.
  • the reaction was worked up by washing the resin with PBS and 1:1 PBS:MeCN (500 ⁇ L each). The cap washed replaced and elute buffer added (300 ⁇ L). The cartridge as vortexed for 30 seconds and agitated for 10 minutes. The cap was removed and the elute buffer was collected. Finally, the DNA was isolated by ethanol precipitation and analyzed by HPLC MS.
  • the quality of P2S5 is important to achieving excellent conversion.
  • the reaction was cooled to ambient temperature and a mixture of MTB E/hexanes (1:1, 0.5 L, 8.3 v.) was added. The resulting mixture was washed with water (3 x 200 mL). Note: The use of hexanes alone caused the product to oil out during work up.
  • the organic phase was concentrated to -150 mL, solids formed during concentration. Methanol (150 mL) was added and the resulting mixture was concentrated to -150 mL. Hexanes (75 mL) was added, then water (100 mL) was added slowly over 25 minutes. The resulting slurry was agitated for 1.5 hours.
  • the batch was filtered and the reactor rinsed with methanol/water (1 : 1, 90 mL), and then hexanes (2 x 60 mL), providing PFP P(V) salt as a white solid (121 g, 81%).
  • (+)-cis-limonene oxide 50 mL was hydrogenated with a balloon of H 2 gas using Pd/C (500 mg, 10%, Johnson Matthey) as a catalyst in ethanol (200 mL). After stirring overnight at room temperature, the crude reaction was filtered through a pad of silica gel to remove the catalyst, after concentration, crude product was obtained as a clear liquid that was used directly in the next reaction.
  • Pd/C 500 mg, 10%, Johnson Matthey
  • the crude reaction was concentrated to remove ⁇ 50-100 mL of solvent (Note: crystals began to form). Water (250 mL, 2.5 v.) was added followed by heptane (250 mL, 2.5 v.) and the reaction was cooled to 5 °C and stirred for 30 minutes. The crude product was collected by filtration, washed with water followed by hexane. Yielding intermediate 1 as a as a white solid (75 g, d.r >99: 1). The crude solids were mixed with DCM (400 mL) (Note: slurry with water layer on top). The water layer was discarded, and the slurry was concentrated to -100 mL.
  • ⁇ 2 -dG was prepared according to General Procedure 1 using dG(Pya) (SI-7) (5.00 g, 7.69 mmol). Purification by SFC (Waters Torus DIOL column 5 mm, 19x160 mm, 100 mL/min, 22-30% MeOH in CO 2 over 3 min 120 bar backpressure at 40 °C) afforded compound ⁇ 2 -dG (0.800 g, 13%).
  • DNA 61 was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 60 was loaded onto two tubes each containing 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum. These tubes were manipulated in parallel.
  • DBU 450 mM, 18 ⁇ L
  • the reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes.
  • the reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 ⁇ L) and 1:1 MeCN:PBS (500 ⁇ L).
  • Elute buffer 300 ⁇ L was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes.
  • the DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation.
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (- 20°C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC- MS analysis was performed (Fig. 10).
  • Compound 61 was synthesized by General Procedure 2 and isolated crude after ethanol precipitation.
  • DNA 61 was dissolved to 300 mM in PBS.
  • Solid BSA was dissolved in PBS (2.5 mg/mL, ⁇ 40 ⁇ M). These two solutions were combined in a PCR tube (25 ⁇ L of each for a reaction volume of 50 ⁇ L) and the resulting reaction mixture was incubated in a thermocycler for 4 hours.
  • the crude ligation solution was diluted to 0.5 mg/mL with respect to BSA and injected for intact protein analysis. Deconvolution across the entire mass peak showed no detectible unmodified BSA remaining. (Fig. 11)
  • DNA SI-40 was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 63 was loaded onto two tubes containing 100 ⁇ L of equilibrated resin. This loaded resin was washed with DMA (500 ⁇ L twice) and dry THF (500 ⁇ L three times) and dried under vacuum. These tubes were manipulated in parallel.
  • DBU 450 mM, 18 ⁇ L
  • the reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes.
  • the reaction was worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 ⁇ L) and 1:1 MeCN:PBS (500 ⁇ L).
  • Elute buffer 300 ⁇ L was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes.
  • the DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation.
  • Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 ⁇ L, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube ( ⁇ 1000 ⁇ L) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed. (Fig. 12)
  • Antibody (h38C2 IgGl) in storage solution was buffer exchanged into PBS by amicon spin filter (30 kDA) and concentrated to 25 ⁇ M ( ⁇ 5 mg/mL). This solution was used to dissolve (50 ⁇ L) solid DNA 64 to a presumptive concertation of 300 ⁇ M.
  • a second tube containing 50 ⁇ L of Antibody was aliquoted and taken through an identical process to serve as a positive control. Dissolving 64 with antibody solution was performed by vortexing the solution. This reaction mixture was incubated at 37 °C for 8 hours to produce DNA- Antibody conjugate 65. The crude reactions mixture was analyzed for conjugation efficiency as previously described by Rader et. al 3
  • the methodol assay for conjugation confirmation was performed as described by Rader and co-workers. Briefly, aliquots (12.5 ⁇ L) of the reaction and control solutions were diluted (0.2 mg/mL relative to original antibody concentration) in PBS to a final volume of 310 ⁇ L. Each sample were dispensed (98 ⁇ L) in triplicate into a black 96-well plate. Three blank wells, containing PBS were also dispensed (98 ⁇ L) into the black plate. A plate reader was prepared, the wavelength of excitation ( ⁇ ext) was set to 330 nm and wavelength of emission ( ⁇ em ) was set to 452 nm. The instrument was programed to record every minute for 60 minutes and shake the plate in between.

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Abstract

Provided herein are methods for site selective chemical modification of an oligonucleotide comprising immobilizing the oligonucleotide on an inert resin to form oligonucleotide-resin complex; and contacting the oligonucleotide-resin complex with a phosphorus (V) based phosphorus (V) reagent in an organic solvent to produce a modified oligonucleotide. Further provide herein are methods of targeting delivery of an oligonucleotide therapeutic to a specific location in the patient body, and/or improving stability and half life of an oligonucleotide therapeutic by using modified oligonucleotides.

Description

SYNTHETIC ELABORATION OF NATIVE DNA BY RASS (SENDR)
[0001] This application claims the benefit of priority to U.S. Provisional Application 63/005,699, filed April 6, 2020, which is incorporated by reference herein in their entirety.
Statement Regarding Federally Sponsored Research Or Development.
[0002] This invention was made with government support under grant numbers GM132787, GM-118176, GM-136286, UL1 TR002551 and award number TL1 TR002551 awarded by the National Institutes of Health. The United States government has certain rights in the invention.
Sequence Listing
[0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 5, 2021, is named SENDR-Sequence_listing_ST25.txt and is 2,176 bytes in size.
Field of the Invention
[0004] The present disclosure relates to the fields of chemical biology and therapeutics, and more specifically methods of modifying oligonucleotides.
Background of the Invention
[0005] The background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0006] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. [0007] Oligonucleotide conjugates are often used in the fields of chemical biology, biophysics, and diagnostics. Indeed, oligonucleotide conjugate-based technology has provided the basis for many modem technological advances. For example, DNA-PAINT conjugates enable transforming super resolution microscopy, and TaqMan PCR probes have revolutionized precision diagnostics.
[0008] Hybridization probes operate through an exquisite molecular recognition ability that a single strand of DNA displays towards its complimentary sequence. The ability for DNA to take on defined inter and intramolecular conformations allows for another dimension of selectivity. Visualizing these molecular interactions usually requires the incorporation of a fluorophore or radioactive moiety. It is this foundation that allows for ubiquitous biochemical and diagnostic techniques such as Southern and Northern Blotting, molecular beacons, and TaqMan qPCR.
[0009] However, while these techniques are commonly used, they suffer from a variety of disadvantages. For example, DNA-PAINT and TaqMan PCR require custom DNA oligomer conjugates that are precisely functionalized and homogeneous. Chemical synthesis of the requisite oligonucleotide conjugates is costly and time consuming. This can make diagnosis of diseases and laboratory experiments cost prohibitive. For example, a custom TaqMan PCR probe must be bought for every diagnostic test or experiment, making it immensely expensive. Furthermore, as oligonucleotide tagging becomes increasingly popular, multiplexed experiments could become even more time consuming and cost prohibitive if thousands of chemically synthesized and modified oligonucleotides are required.
[0010] Recently oligonucleotide therapeutics have become popular because they have the potential to therapeutically regulate essentially any gene of interest at the DNA or RNA level. Their versatility in treating inherited or acquired disorders stems from the ability to induce efficient gene silencing, gene expression or gene editing.
[0011] Despite these advantages, using oligonucleotide as a therapeutic in vivo is challenging because of their unfavorable physicochemical characteristics. For example, oligonucleotides are susceptible to degradation by nucleases in the circulation, suffer from rapid renal clearance, and induce immunostimulatory effects via pattern recognition receptors, resulting in adverse effects. [0012] Thus, there remains a need in the art for novel ways to economically, simply, and quickly build oligonucleotide conjugates from native (unprotected) substrates or chemically modified oligonucleotide substrates. There also remains a need in the art for oligonucleotide therapeutics that overcome the disadvantages outlined above.
Summary of The Invention
[0013] The inventors have now disclosed techniques for selectively and effectively targeting and tagging the hydroxyl group at the 5’ and/or 3’ end of an oligonucleotide (for example, a DNA, or RNA) with a Phosphorus(V) (“P(V)”) based reagent, to thereby incorporate a small molecule handle linked via P(V) bond into the oligonucleotide. The resulting modified oligonucleotide conjugate can be a stable, nuclease resistant biomimetic having a P(V) linkage. The exquisite selectivity and rapid reaction time of the methods and disclosed herein allows for site selective chemical modification of unprotected, native, DNA for the production of DNA based florescent probes, diagnostics, conjugates or therapeutics. The oligonucleotide may be an unprotected or native. This method of oligonucleotide modification has been named SENDR by the inventors (Synthetic Elaboration of Native DNA by RASS).
[0014] In one embodiment, disclosed herein is a method for site selective chemical modification of an oligonucleotide comprising: immobilizing the oligonucleotide on a solid support to form oligonucleotide-solid support complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-solid support complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the solid support.
[0015] The site-selective chemical modification of the oligonucleotide may comprise modification at 5’ hydroxyl group or 3’ hydroxyl group, or both 5’ and 3’ hydroxyl groups. In this method, the phosphorus (V) reagent is contemplated to have a chemical structure
, wherein X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’, R’
Figure imgf000005_0001
is at each occurrence are independently selected from the group consisting hydrogen, C1to C10 alkyl, C1 to C10 heteroalkyl, C1 to C10 cycloalkyl, C1 to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl, Y and Z are each independently selected from the group consisting of O, S, and N, R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein: any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle.
[0016] In some embodiments, the phosphorus (V) reagent has the chemical structure
Figure imgf000006_0001
[0017] The oligonucleotide may be a DNA, RNA, LNA, MPO, PMO, or derivations and chimeric mixtures thereof. The modified oligonucleotide is preferably modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group. The oligonucleotide may be structured aptamer, primer or hybridization probe, or a library thereof. In some cases, the oligonucleotide is further conjugated to a protein to form a oligonucleotide-protein conjugate. The protein is preferably a therapeutic antibody. In some embodiments, the oligonucleotide is modified at multiple specific locations.
[0018] The method disclosed herein may further comprise eluting the modified oligonucleotide from the solid support by washing the oligonucleotide-solid support complex with an aqueous buffer solution. The P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes. The modified oligonucleotide may further conjugate with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins. The biorthogonal chemistry may comprise Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide formation, amide coupling, proximity photoaffinity labeling, hydrazone ligation, and CuAAC reaction. In some embodiments, the oligonucleotide comprises unnatural nucleotides. In some cases, the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
[0019] In another embodiment, disclosed herein is a method of increasing the half-life of an oligonucleotide therapeutic in-vivo, the method comprising: immobilizing the oligonucleotide on an inert solid support to form oligonucleotide-solid support complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-solid support complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the inert solid support, wherein the site specific P(V) reagent modification increases stability and the half-life of the oligonucleotide therapeutic in vivo. In yet another embodiment, disclosed herein is a method of targeting delivery of an oligonucleotide therapeutic to a specific location in the body of a patient, the method comprising: attaching a sugar or lipid to a therapeutic oligonucleotide by the method of claim 1 to thereby produce a modified therapeutic oligonucleotide; and administering the modified therapeutic oligonucleotide to the patient for targeted delivery to a specific location in the patient body.
[0020] In each of the above embodiments, the site-selective chemical modification of the oligonucleotide comprises modification at 5’ hydroxyl group, or 3’ hydroxyl group or both 5’ and 3’ hydroxyl groups. In one embodiment, the phosphorus (V) reagent has a chemical
Figure imgf000008_0001
structure , wherein X is a leaving group having the formula O-R’, S-R’, N-R’,
C-R’, R’ is at each occurrence are independently selected from the group consisting hydrogen, C1 to C10 alkyl, C1 to C10 heteroalkyl, C1 to C10 cycloalkyl, C1 to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1to C10 aralkyl, Y and Z are each independently selected from the group consisting of O, S, and N, R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxycarbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein: any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle.
[0021] In one embodiment, the oligonucleotide therapeutic is a DNA, RNA, LNA, MPO, PMO, or derivations or chimeric mixtures thereof. In one embodiment, the modified therapeutic oligonucleotide is modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group. In one embodiment, the therapeutic oligonucleotide is a structured aptamer or hybridization probe. In one embodiment, the therapeutic oligonucleotide is further conjugated to a protein to form an oligonucleotide-protein conjugate. In one embodiment, the protein is a therapeutic antibody. In one embodiment, the oligonucleotide is modified at multiple specific locations. In one embodiment, the method further comprises eluting the modified oligonucleotide from the solid support by washing the oligonucleotide-solid support complex with an aqueous buffer solution. In one embodiment, the P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes. In one embodiment, the modified oligonucleotide further conjugates with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins. In one embodiment, the biorthogonal chemistry comprises Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffinity labeling, hydrazone ligation, and CuAAC reaction. In one embodiment, the oligonucleotide comprises unnatural nucleotides. In one embodiment, the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
[0022] In another embodiment, disclosed herein is a kit for DNA modification, comprising:
(a) P(V) reagent having the formula
Figure imgf000009_0001
, wherein X= O-R’, S-R’, N-R’, C-R’, R’ is at each occurrence are independently selected from the group consisting hydrogen, C1to C10 alkyl, C1 to C10 heteroalkyl, C1 to C10 cycloalkyl, C1 to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl, Y and Z are each independently selected from the group consisting of O, S, and N, R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide and amino, wherein: any two of R1, R2, R3 , and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle; (b) l,8-Diazabicyclo[5.4. 0]undec-7-ene (DBU); (c) Dry Tetrahydrofuran (THF); (d) Dimethylacetamide (DMA); (e) Elution buffer, and (f) solid support in fitted cartridge. In some embodiments, the elution buffer comprises 1M NaC104, 20% MeOH, 40 mM Tris pH 8.5.
[0023] In another embodiment, disclosed herein is a method of diagnosing a disease in a patient in need thereof, comprising: obtaining a tissue sample, blood sample, or body fluid swab from the patient; extracting oligonucleotides from the patient sample; labeling and/or modifying oligonucleotide with by the method of claim 1 that may be cognate to the disease of interest; performing PCR reaction on the labeled and/or modified oligonucleotide; and analyzing the labeled and/or modified oligonucleotide to diagnose the disease. In one embodiment, the disease is an infectious disease. In one embodiment, the disease is a bacterial or viral disease, and the patient is diagnosed with the disease if the analyzed labeled and/or modified oligonucleotide comprises the bacterial or viral DNA. In one embodiment, the disease is a tumor, and the patient is diagnosed with the tumor if the analyzed labeled and/or modified oligonucleotide comprises cancer specific DNA mutations.
[0024] In another embodiment, the reactions disclosed herein may be used to create libraries or combinatorial libraries of ligated oligonucleotides linked through two chemical handles incorporated on the termini of the oligo. In some embodiments this strategy could be sued to create circularized oligonucleotides. In some embodiments this strategy could be used to assemble synthetic or natural genes or gene fragments
[0025] Various objects, features, aspects, and advantages will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing in which like numerals represent like components.
Brief Description of The Drawing
[0026] Fig. 1 depicts an exemplary modification of native DNA. (A) prior art method of enzymatic stochastic labeling of DNA. (B) prior art method of chemical stochastic labeling of DNA. (C) Chemical modification of DNA, Phosphoramidate formation. (D) chemical modification of oligonucleotide using the SENDR method disclosed herein.
[0027] Fig. 2 depicts exemplary ψ-modules synthesized for this study.
[0028] Fig. 3 depicts P(V) based DNA modification. (A) SENDR enabled DNA modification. (B) Optimization of the coupling step. (C) Substrate scope. Conversions based on HPLC integration of a total absorbance signal at 260 nm. Unless otherwise noted, standard reaction conditions were applied; ψ-module (150 mM), DBU (450 mM), in dry MeCN (250 μL), 60 min, r.t. while adsorbed to Strata XL-A. a75 mM PSI and 225 mM DBU, b45 °C,c37 °C. d200 mM PSI at 50 °C. e300 mM PSI at 37 °C. DNA loading (adsorption step) performed in PBS. Resin washed with DMA (x2) and THF (x3). Resin dried under a vacuum 2 h. Elution was performed using elution buffer 1 M NaC104, 40 mM Tris pH 8.5, 20% MeOH. In-situ protocol
[0029] Fig. 4 depicts exemplary downstream synthetic manipulations of SENDR-derived DNA-small molecule hybrids (ligated at 3' or 5')
[0030] Fig. 5 depicts exemplary SENDR compatibility with PS DNA and lager structured oligomers. (Top) SENDR compatibility with PS DNA. (Bottom) SENDR compatibility with larger structured oligomers. Standard reaction conditions were applied; ψ-module (150 mM), DBU (450 mM), in dry MeCN (250 μL), 60 min, 37 °C while adsorbed to Strata XL-A.
[0031] Fig. 6 depicts exemplary SENDR aptamer modification. (A) Standard reaction conditions were applied; ψ-module (150 mM), DBU (450 mM), in dry MeCN (250 μL), 60 min, r.t. while adsorbed to Strata XL-A. (B) Direct incorporation of electrophiles into aptamers and their inhibition of protein targets. The reaction conditions that were applied: ψ-module (150 mM), DBU (450 mM), in dry MeCN (250 μL), 60 min, 37 °C, while adsorbed to Strata XL-A.
[0032] Fig. 7 depicts exemplary SENDR on biosynthetically derived DNA. Scheme representing the biosynthetic steps to produce the COVID-19 N gene amplicon (59). HPLC chromatogram of the SENDR reaction. Deconvoluted mass spectrum of the starting material peak and the product peak.
[0033] Fig. 8 depicts exemplary SENDR enabled DNA-protein conjugation. (A) DNA-BSA conjugation (ESI-TOF mass spectra of starting material and product). (B) cTegsedi-DVD conjugation (SDS PAGE and catalytic methodol fluorescence assay including control experiments).
[0034] Fig. 9 depicts exemplary creation of dual labeled DNA probes. (A) The synthesis of a TaqMan probe for RNaseP. (B) Synthesis of the COVID 19 qPCR panel of probes.
[0035] Fig. 10 illustrates the LCMS Characterization of 61
[0036] Fig. 11 illustrates an embodiment of analysis of DNA-antibody (h38C2 IgG1) conjugation by the methods disclosed herein.
[0037] Fig. 12 illustrates the LCMS Characterization of SI-40
[0038] Fig. 13 illustrates the LCMS Characterization of 64.
[0039] Fig. 14 illustrates one embodiment of the methodol assay as used herein.
[0040] Fig. 15 illustrates SDS Page analysis of an antibody-DNA construct.
[0041] Fig. 16 illustrates the LCMS Characterization of compound 52. The DNA corresponding to this compound is SEQ ID NO: 10 [0042] Fig. 17 illustrates the LCMS Characterization of compound 53. The DNA corresponding to this compound is SEQ ID NO: 10.
Detailed Description
[0043] The inventors have now discovered a method for non-covalent immobilization of an oligonucleotide, and then chemical modification the oligonucleotide at the native terminal hydroxyl groups. This new site-specific oligonucleotide modification approach results in reliable and specific incorporation of a chemical moiety in the 5’ and/or 3’ end of a nucleic acid.
[0044] Although many chemical and biochemical methods exist for the stochastic labeling of native DNA, all methods produce non homogenous end products that contain modified bases determined stochastically (Figure 1 A and B). In some cases, DNA with randomly incorporated labels can be useful but unlocking the total power of DNA as a molecular recognition probe requires site selective incorporation of the desired tag. Towards this end, Chu et al (Nucleic Acids Res, 1983, 11, 6513-6529) reported a method for the site-specific modification of native DNA. The Chu disclosure relies on a water soluble carbodiimide (EDC), imidazole and simple amines to furnish a phosphoramidate linkage between native DNA bearing a 5’ phosphate group and an amine of interest. However, when the inventors tried this method for modifying an oligonucleotide, the process and the resulting products provided to be unreliable, and the inventors obtained a product that was littered with many EDC adducts, even after multiple attempts (Figure 1C).
[0045] To remedy this, the inventors employed the P(V) based reagent chemistry as described in W02019200273A1 and Knouse et al., Science 361, 1234-1238 (2018), all of which are incorporated by reference herein. This method constructs phosphorus based linkages between two alcohols in a chemo selective fashion (Figure ID).
[0046] The inventors found that the compounds and P(V) reagents, disclosed in the above two disclosures, employ in dry acetonitrile, as P(V) reagents readily hydrolyze in water. On the other hand, due to the highly charged nature of the DNA phosphate backbone, native DNA is insoluble in most organic solvents and usually requires significant water content to solubilize it in an aqueous system. These irreconcilable facts precluded the simple adaptation of P(V) for the purposes of site-specific DNA labeling. [0047] The inventors then decided to incorporate the Reversible Adsorption of Solid Support (RASS) method performing chemistry on-DNA, as disclosed in PCT /US2019/048570, which is incorporated by reference herein in its entirety, including the drawings. Reversible Adsorption of Solid Support (RASS) is a process which allows for the adsorption of biomacromolecules onto a solid support to facilitate their transfer into solvents or reaction paradigms that would previously be considered incompatible. In this iteration, DNA is adsorbed to a polystyrene based cationic support, through a simple mixing procedure, and the solvent exchanged (by simple washing and drying) into near anhydrous conditions to perform water incompatible reactions.
[0048] With this reaction modality in hand, the inventors applied P(V) chemistry for the site- specific labeling of native oligonucleotide. It should be recognized that P(V) reagents include PSI, PS2, PO2, Rac-PSI, or PI. A variety of P(V) ψ-loaded reagents (ψ modules, Fig. 2) were prepared. Model DNA 1 and 2, each contain a single modifiable terminal hydroxyl group and a single terminal phosphate which acts as a native protecting group (Figure 3A). As would be known to a skilled artisan, the DNA sequences in Model DNA 1 and 2 are for illustrative purposes only, and any DNA or RNA sequences may be used in the methods disclosed herein.
[0049] The inventors found that, while DNA could be readily adsorbed to the support, initial attempts failed or provided low yields of the modified product. During these studies, the inventors washed the resin bound DNA at least three times with acetonitrile prior to performing the reaction.
[0050] The inventors postulated that these reactions may have failed due to residual water inactivating the PSI reagents upon the addition of DBU. Thereupon, a simple yet stringent drying protocol was devised, in which resin bound DNA was washed with both DMA and THF before drying under vacuum. With this protocol in hand, optimization of the desired reaction proceeded smoothly. The optimal conditions were identified and produced singly labeled products in good yields at both the 5’ and 3’ position, with no by-products. The labeling position was confirmed by MS fragmentation. The conditions were also found to be sequence independent.
[0051] Accordingly, one aspect of the present disclosure provides methods for site selective chemical modification of an oligonucleotide. The method comprises immobilizing the oligonucleotide on an inert resin to form oligonucleotide-resin complex and contacting the oligonucleotide-resin complex with a phosphorus (V) based phosphorus-sulfur incorporation (PSI) reagent in an organic solvent to produce a modified oligonucleotide. Preferably, the oligonucleotides are DNA or RNA, natural or unnatural.
[0052] Throughout this disclosure, the terms “solid support,” “resin,” and “inert resin” are used interchangeably. The solid support or stationary phase is the phase that is either a solid or liquid particle surface on which the components of the mixture to be separated is absorbed or adsorbed selectively. The support remains stationary while the other phases move. Preferably, the solid support used herein comprise of porous materials, thus allowing the attachment of components during adsorption. Many different types of solid support are contemplated in this present disclosure, such as gel beads, thin paper preferably thin uniform paper, silica, glass, gases, or liquid components. The exact nature of the solid support used to practice the methods of this disclosure would depend on the nature of the components to be separated.
[0053] It should be noted that, throughout the disclosure, the terms “nucleic acid” “oligonucleotide” and/or “polynucleotide” are used interchangeably herewith and includes any nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)), analogs thereof, and polymers thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-gly cosides or C-gly cosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as “intemucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. The nucleic acids, oligonucleotides, and polynucleotides as used herein may be naturally occurring, synthetically prepared, or biosynthetically prepared. [0054] The terms “nucleic acid” “oligonucleotide” and/or “polynucleotide” includes Antisense oligonucleotides or oligomers (ASOs). ASOs are short synthetic nucleic acid analogs that are being increasingly used as therapeutics. These oligonucleotides which bind RNA targets through Watson-Crick-Franklin base pairing, result in reduced gene expression or alterations in RNA processing, depending on their design and mode of action for the therapeutic.
[0055] Natural nucleic acids are rapidly degraded by endogenous nucleases in vivo. Therefore, it is important to chemically modify oligonucleotides to increase their resistance to various nucleases, as well as their binding affinity to RNA targets. Phosphorothioate (PS) is a first- generation modification, in which a non-bridging oxygen is replaced by a sulfur atom in the phosphate backbone. Although PS modification significantly increases half-life of the oligonucleotide in vivo, reflecting increased protein binding and increased resistance to nuclease cleavage, it reduces the affinity for the target RNA; moreover, PS-modified DNA oligonucleotides support RNase H activity. Second-generation modifications, such as 2'-O- methyl (2'-O-Me), 2'-O-methoxy ethyl (MOE), constrained ethyl (cEt), and locked nucleic acid (LNA) further increase metabolic stability of oligonucleotides, as well as the binding affinity for RNA in vivo, conferring enhanced drug-like properties. The present disclosure provides methods and techniques for making such modified oligonucleotides for increased stability (half-life).
[0056] The terms “nucleic acid” “oligonucleotide” and/or “polynucleotide” as used herein further contemplates Phosphordiamidate (PDA) morpholino oligomers (PMOs). PMOs replace the pentose sugar with a morpholine ring, and the phosphate with a neutral PDA linkage. PMOs exhibit similar binding affinity to RNA as DNA, have significantly enhanced metabolic stability as well as low protein binding due to the uncharged backbone, and do not support RNase H activity. The low protein binding feature also makes high-dose PMOs safer in vivo, compared to other chemistries.
[0057] The terms “nucleic acid” “oligonucleotide” and/or “polynucleotide” as used herein further contemplates Locked Nucleic Acid (LNA) oligomeric compounds (also referred herein as LNA oligomers or LNA oligonucleotides). An LNA oligomer comprises at least one LNA nucleoside, such as a nucleoside which comprises a covalent bridge (also referred to a radical) between the 2' and 4' position (a 2'-4' bridge). LNA nucleosides are also referred to as “bicyclic nucleosides”. The LNA oligomer is typically a single stranded oligonucleotide. In some embodiments the LNA oligomer comprises or is a gapmer. In some embodiments the LNA oligomer comprises or is a mixmer. In some embodiments the LNA oligomer comprises or is a totalmer. In some embodiments, the nucleoside analogues present in the oligomer are all LNA, and the oligomer may, optionally further comprise RNA or DNA, such as DNA nucleosides (e.g. in a gapmer or mixmer).
[0058] Furthermore, the terms “nucleic acid” “oligonucleotide” and/or “polynucleotide” as used herein also contemplates Methylphosphonate (MP) oligonucleotides (MPOs). MPOs are metabolically stable analogs of conventional DNA or RNA and contains a methyl group in place of one of the non-bonding phosphoryl oxygens. MPOs are highly resistant to metabolic breakdown in biological systems. Unlike natural phosphodiester oligonucleotides, MPOs contain chiral linkages.
[0059] The terms “nucleic acid” “oligonucleotide” and/or “polynucleotide”, as used herein, are polymers of nucleosides joined, generally, through phosphodiester linkages, although alternate linkages, such as phosphorothioate esters may also be used in oligonucleotides. A nucleoside consists of a purine (adenine (A) or guanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U), or derivative thereof) base bonded to a sugar. The four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine. A nucleotide is a phosphate ester of a nucleoside.
[0060] Many derivatives of nucleosides and nucleobases are known in the art and each of them are explicitly contemplated herein. In some embodiments, the nucleoside comprises a naturally-occurring nucleobase, such as adenine, guanine, cytosine, uridine, thymine, 5 -methyl cytosine, etc. In other embodiments, the nucleoside comprises other natural nucleobases, as well as modified nucleobases, such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5 -halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine. Further naturally- and non-naturally-occurring nucleobases include those disclosed in U.S. Patent No. 3,687,808 (Merigan et al); in Sanghvi, in Antisense Research and
Application, Chapter 15, S. T. Crooke and B. Lebleu, Eds., CRC Press, 1993; in Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613-722 (particularly, pages 622 and
623); in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz, Ed.,
John Wiley & Sons, 1990, pages 858-859; in Zhang, et al, Nature, 2017, 551, 644-647 (hydrophobic bases); in Feldman and Romesberg, Acc. Chem. Res. 2018, 51, 394-403; and in Cook, Anti-Cancer Drug Design, 1991, 6, 585-607, each of which is hereby incorporated by reference in its entirety.
[0061] Other examples of modifications of nucleosides and nucleobases described herein include, but are not limited to the following: 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6- methyladenosine; N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1- methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2- methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6- acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6-methyl-N6- threonylcarbamoyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine; Nl- methyl-adenosine; N6, N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio- adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2- (halo)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy- adenosine triphosphate; 2'-azido-2'- deoxy- adenosine triphosphate; 2'-deoxy-2'-a-aminoadenosine triphosphate; 2'-deoxy-2'-a- azidoadenosine triphosphate; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkynyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8- (amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-deazaadenosine triphosphate; 2'fluoro-N6-Bz- deoxyadenosine triphosphate; 2'-methoxy-2-amino- adenosine triphosphate; 2'0-methyl-N6- Bz-deoxyadenosine triphosphate; 2'-a-Ethynyladenosine triphosphate; 2-aminoadenine; 2- aminoadenosine triphosphate; 2-amino- adenosine triphosphate; 2'-a-trifluoromethyladenosine triphosphate; 2-azidoadenosine triphosphate; 2'-b-ethynyladenosine triphosphate; 2- bromoadenosine triphosphate; 2'-b-trifluoromethyladenosine triphosphate; 2-chloroadenosine triphosphate; 2'-deoxy-2',2'-difluoroadenosine triphosphate; 2'-deoxy-2'-a-mercaptoadenosine triphosphate; 2'-deoxy-2'-a-thiomethoxyadenosine triphosphate; 2'-deoxy-2'-b- aminoadenosine triphosphate; 2'-deoxy-2'-b-azidoadenosine triphosphate; 2'-deoxy-2'-b- bromoadenosine triphosphate; 2'-deoxy-2'-b-chloroadenosine triphosphate; 2'-deoxy-2'-b- fluoroadenosine triphosphate; 2'-deoxy-2'-b-iodoadenosine triphosphate; 2'-deoxy-2'-b- mercaptoadenosine triphosphate; 2'-deoxy-2'-b-thiomethoxyadenosine triphosphate; 2- fluoroadenosine triphosphate; 2-iodoadenosine triphosphate; 2-mercaptoadenosine triphosphate; 2-methoxy -adenine; 2-methylthio-adenine; 2-trifluoromethyladenosine triphosphate; 3-deaza-3-bromoadenosine triphosphate; 3-deaza-3-chloroadenosine triphosphate; 3-deaza-3-fluoroadenosine triphosphate; 3-deaza-3-iodoadenosine triphosphate; 3-deazaadenosine triphosphate; 4'-azidoadenosine triphosphate; 4'-carbocyclic adenosine triphosphate; 4'-ethynyladenosine triphosphate; 5'-homo-adenosine triphosphate; 8-aza- adenosine triphosphate; 8-bromo-adenosine triphosphate; 8-Trifluoromethyladenosine triphosphate; 9-deazaadenosine triphosphate; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7- deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8- aza-adenine, 7-deaza-2-aminopurine; 2-thiocytidine; 3-methylcytidine; 5-formylcytidine; 5- hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 5,2'-O- dimethylcytidine; 5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine; N4- acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4- methy Icy ti dine; 5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine; 2- (thio)cytosine; 2'-amino-2'-deoxy- cytidine triphosphate; 2'-azido-2'-deoxy- cytidine triphosphate; 2'-deoxy-2'-a-aminocytidine triphosphate; 2'-deoxy-2'-a-azidocytidine triphosphate; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5- (alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5- bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1 -methyl- 1-deaza-pseudoisocyti dine; 1-methyl- pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl- cytidine; 4-methoxy-l-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-l- methyl- 1-deaza-pseudoisocyti dine; 4-thio-l-methyl-pseudoisocytidine; 4-thio- pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine; (E)-5-(2-bromo-vinyl)cytidine triphosphate; 2,2'-anhydro-cytidine triphosphate hydrochloride; 2'fluor-N4-Bz-cytidine triphosphate; 2'fluoro-N4-acetyl-cytidine triphosphate; 2'-O-methyl-N4-acetyl-cytidine triphosphate; 2'0-methyl-N4-Bz-cytidine triphosphate; 2'-a- ethynylcytidine triphosphate; 2'-a-trifluoromethylcytidine triphosphate; 2'-b-ethynylcytidine triphosphate; 2'-b-trifluoromethylcytidine triphosphate; 2'-deoxy-2',2'-difluorocytidine triphosphate; 2'-deoxy-2'-a-mercaptocytidine triphosphate; 2'-deoxy-2'-a-thiomethoxycytidine triphosphate; 2'-deoxy-2'-b-aminocytidine triphosphate; 2'-deoxy-2'-b-azidocytidine triphosphate; 2'-deoxy-2'-b-bromocytidine triphosphate; 2'-deoxy-2'-b-chlorocytidine triphosphate; 2'-deoxy-2'-b-fluorocytidine triphosphate; 2'-deoxy-2'-b-iodocytidine triphosphate; 2'-deoxy-2'-b-mercaptocytidine triphosphate; 2'-deoxy-2'-b-thiomethoxycytidine triphosphate; 2'-O-methyl-5-(l-propynyl)cytidine triphosphate; 3'-ethynylcytidine triphosphate; 4'-azidocytidine triphosphate; 4'-carbocyclic cytidine triphosphate; 4'- ethynylcytidine triphosphate; 5-(l-propynyl)ara-cytidine triphosphate; 5-(2-chloro-phenyl)-2- thiocytidine triphosphate; 5-(4-amino-phenyl)-2-thiocytidine triphosphate; 5-aminoallyl- cytidine triphosphate; 5-cyanocytidine triphosphate; 5-ethynylara-cytidine triphosphate; 5- ethynylcytidine triphosphate; 5'-homo-cytidine triphosphate; 5 -methoxy cytidine triphosphate; 5-trifluoromethyl-cytidine triphosphate; N4-amino-cytidine triphosphate; N4-benzoyl-cytidine triphosphate; pseudoisocytidine; 7-methylguanosine; N2,2'-O-dimethylguanosine; N2- methylguanosine; wyosine; l,2'-O-dimethylguanosine; 1 -methylguanosine; 2'-O- methylguanosine; 2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine; 2'-O- ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; archaeosine; methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6- thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; Nl-methyl-guanosine; a-thio- guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy- guanosine triphosphate; 2'-Azido-2'-deoxy- guanosine triphosphate; 2'-deoxy-2'-a-aminoguanosine triphosphate; 2'- deoxy-2'-a-azidoguanosine triphosphate; 6-(alkyl)guanine;; 6-methyl-guanosine; 7- (alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8- (thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N-(methyl)guanine; 1- methyl-6-thio-guanosine; 6-methoxy -guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7- deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo- guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-me- guanosine triphosphate; 2'fluoro-N2-isobutyl-guanosine triphosphate; 2'0-methyl-N2-isobutyl- guanosine triphosphate; 2'-a-ethynylguanosine triphosphate; 2'-a-trifluoromethylguanosine triphosphate; 2'-b-ethynylguanosine triphosphate; 2'-b-trifluoromethylguanosine triphosphate; 2'-deoxy-2',2'-difluoroguanosine triphosphate; 2'-deoxy-2'-a-mercaptoguanosine triphosphate; 2'-deoxy-2'-a-thiomethoxyguanosine triphosphate; 2'-deoxy-2'-b-aminoguanosine triphosphate; 2'-deoxy-2'-b-azidoguanosine triphosphate; 2'-deoxy-2'-b-bromoguanosine triphosphate; 2'-deoxy-2'-b-chloroguanosine triphosphate; 2'-deoxy-2'-b-fluoroguanosine triphosphate; 2'-deoxy-2'-b-iodoguanosine triphosphate; 2'-deoxy-2'-b-mercaptoguanosine triphosphate; 2'-deoxy-2'-b-thiomethoxyguanosine triphosphate; 4'-azidoguanosine triphosphate; 4'-carbocyclic guanosine triphosphate; 4'-ethynylguanosine triphosphate; 5'- homo-guanosine triphosphate; 8-bromo-guanosine triphosphate; 9-deazaguanosine triphosphate; N2-isobutyl-guanosine triphosphate; 1-methylinosine; inosine; l,2'-O- dimethylinosine; 7-methylinosine; 2'-O-methylinosine; epoxyqueuosine; galactosyl- queuosine; mannosylqueuosine; queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5- hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine; dihydrouridine; pseudouridine; l-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1- methylpseduouridine; 1 -ethyl-pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O- dimethyluridine; 3-methyl-pseudo-uridine triphosphate; 4-thiouridine; 5- (carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2'-O- dimethyluridine; 5,6-dihydro-uridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O- methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5- carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O- methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyluridine; 5-carbamoylmethyluridine triphosphate; 5- methoxycarbonylmethyl-2'-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5- methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5- methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5- methylaminomethyluridine; 5-methyldihydrouridine; 5-oxyacetic acid- uridine triphosphate; 5-oxyacetic acid-methyl ester-uridine triphosphate; N1 -methyl-pseudo-uracil; N1 -ethyl- pseudo-uracil; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3- carboxypropyl)-uridine triphosphate; 5-(iso-pentenylaminomethyl)- 2-thiouridine triphosphate; 5-(iso-pentenylaminomethyl)-2'-O-methyluridine triphosphate; 5-(iso- pentenylaminomethyl)uridine triphosphate; 5-propynyl uracil; a-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; l-(aminoalkylamino-carbonylethylenyl)-2- (thio)-pseudouracil; l-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine triphosphate; 1- methyl-3-(3-amino-3-carboxypropyl)pseudo- uridine triphosphate; 1 -methyl-pseudo- uridine triphosphate; 1 -ethyl-pseudo- uridine triphosphate; 2 (thio)pseudouracil; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl, 2'amino, 2'azido, 2'fluro- guanosine; 2'-amino-2'-deoxy-uridine triphosphate; 2'-azido-2'-deoxy- uridine triphosphate; 2'- azido-deoxyuridine triphosphate; 2 deoxy uridine; 2 fluorouridine; 2'-deoxy-2'-a- aminouridine triphosphate; 2'-deoxy-2'-a-azidouridine triphosphate; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4-(thio )pseudouracil; 4-thiouracil; 5 (1,3-diazole-l- alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl- methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2- aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5- (alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5- (allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-
(dimethylaminoalkyl)uracil; 5-(guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(l,3-diazole-l- alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5- (methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio )uracil; 5- (methyl) 4 (thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)-2,4(dithio )uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo- uridine; 5-uracil; 6-(azo)uracil; 6-aza-uridine; ally amino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; pseudo-uridine triphosphate- 1-2-ethanoic acid; pseudouracil; 4-thio-pseudo- uridine triphosphate; 1 -carboxymethy 1-pseudouridine; 1 -methyl- 1-deaza-pseudouri dine; 1- propyny 1-uridine; 1-taurinomethyl-l -methyl-uridine; 1 -taurinomethyl-4-thio-uridine; 1- taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio- 1 -methyl- 1-deaza- pseudouridine; 2-thio- 1 -methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio- dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio- pseudouridine; 4-methoxy -pseudouridine; 4-thio-1 -methyl-pseudouridine; 4-thio- pseudouridine; 5-aza-uridine; dihydropseudouridine; (±)l-(2-hydroxypropyl)pseudouridine triphosphate; (2R)-1-(2-hydroxypropyl)pseudouridine triphosphate; (2S)-1-(2- hydroxypropyl)pseudouridine triphosphate; (E)-5-(2-bromo-vinyl)ara-uridine triphosphate; (E)-5-(2-bromo-vinyl)uridine triphosphate; (Z)-5-(2-Bromo-vinyl)ara-uridine triphosphate; (Z)-5-(2-bromo-vinyl)uridine triphosphate; 1 -(2,2,2-trifluoroethyl)-pseudo-uridine triphosphate; 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine triphosphate; 1-(2,2- di ethoxy ethy l)pseudouridine triphosphate; 1 -(2,4,6-trimethylbenzy l)pseudouridine triphosphate; 1-(2,4,6-trimethyl-benzyl)pseudo-uridine triphosphate; 1-(2,4,6-trimethyl- phenyl)pseudo-uridine triphosphate; 1-(2-smino-2-carboxyethyl)pseudo-uridine triphosphate; 1-(2-amino-ethyl)pseudo-uridine triphosphate; 1 -(2 -hydroxy ethy l)pseudouri dine triphosphate; 1-(2-methoxyethyl)pseudouridine triphosphate; 1-(3,4-bis- trifluoromethoxybenzyl)pseudouridine triphosphate; l-(3,4-dimethoxybenzyl)pseudouridine triphosphate; 1-(3-amino-3-carboxypropyl)pseudo-uridine triphosphate; 1-(3-amino- propyl)pseudo-uridine triphosphate; 1-(3-cyclopropyl-prop-2- ynyl)pseudouridinetriphosphate; 1-(4-amino-4-carboxybutyl)pseudo-uridine triphosphate; 1- (4-amino-benzyl)pseudo-uridine triphosphate; 1 -(4-amino-butyl)pseudo-uridine triphosphate; 1-(4-amino-phenyl)pseudo-uridine triphosphate; 1-(4-azidobenzyl)pseudouridine triphosphate; 1-(4-bromobenzyl)pseudouridine triphosphate; 1-(4-chlorobenzyl)pseudouridine triphosphate; 1-(4-fluorobenzyl)pseudouridine triphosphate; 1 -(4-iodobenzyl)pseudouridine triphosphate; 1-(4-methanesulfonylbenzyl)pseudouridine triphosphate; 1-(4- methoxybenzyl)pseudouridine triphosphate; 1 -(4-methoxy-benzyl)pseudo-uridine triphosphate; 1-(4-methoxy-phenyl)pseudo-uridine triphosphate; 1-(4-methyl-benzyl)pseudo- uridine triphosphate; 1-(4-nitro-benzyl)pseudo-uridine triphosphate; 1 (4-nitro-phenyl )pseudo- uridine triphosphate; 1-(4-thiomethoxybenzyl)pseudouridine triphosphate; 1-(4- trifluoromethoxybenzyl)pseudouridine triphosphate; 1-(4- trifluoromethylbenzyl)pseudouridine triphosphate; 1-(5-amino-pentyl)pseudo-uridine triphosphate; 1-(6-amino-hexyl)pseudo-uridine triphosphate; 1,6-dimethyl-pseudo-uridine triphosphate; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)- propionyl] pseudouridine triphosphate; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl } pseudouridine triphosphate; 1-acetylpseudouridine triphosphate; 1-alkyl-6-(1-propynyl)- pseudo-uridine triphosphate; 1-alkyl-6-(2-propynyl)-pseudo-uridine triphosphate; 1-alkyl-6- allyl-pseudo-uridine triphosphate; l-alkyl-6-ethynyl-pseudo-uridine triphosphate; 1 -alky 1-6- homoallyl-pseudo-uridine triphosphate; 1-alkyl-6-vinyl-pseudo-uridine triphosphate; 1- allylpseudouridine triphosphate; 1-aminomethyl-pseudo-uridine triphosphate; 1- benzoylpseudouridine triphosphate; 1-benzyloxymethylpseudouridine triphosphate; 1 -benzyl- pseudo-uridine triphosphate; 1-biotinyl-PEG2-pseudouridine triphosphate; 1- biotinylpseudouridine triphosphate; 1-butyl-pseudo-uridine triphosphate; 1- cyanomethylpseudouridine triphosphate; 1-cyclobutylmethyl-pseudo-uridine triphosphate; 1- cyclobutyl-pseudo-uridine triphosphate; 1-cycloheptylmethyl-pseudo-uridine triphosphate; 1- cycloheptyl-pseudo-uridine triphosphate; 1-cyclohexylmethyl-pseudo-uridine triphosphate; 1- cyclohexyl-pseudo-uridine triphosphate; 1-cyclooctylmethyl-pseudo-uridine triphosphate; 1- cyclooctyl-pseudo-uridine triphosphate; 1-cyclopentylmethyl-pseudo-uridine triphosphate; 1- cyclopentyl-pseudo-uridine triphosphate; 1-cyclopropylmethyl-pseudo-uridine triphosphate; 1 -cyclopropyl-pseudo-uridine triphosphate; 1 -hexyl-pseudo-uridine triphosphate; 1- homoallylpseudouridine triphosphate; 1-hydroxymethylpseudouridine triphosphate; 1 -iso- propyl-pseudo-uridine triphosphate; l-me-2-thio-pseudo-uridine triphosphate; 1-me-4-thio- pseudo-uridine triphosphate; 1-me-alpha-thio-pseudo-uridine triphosphate; 1- methanesulfonylmethylpseudouridine triphosphate; 1 -methoxymethylpseudouridine triphosphate; 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-uridine triphosphate; 1 -methyl-6-(4- morpholino)-pseudo-uridine triphosphate; 1 -methyl-6-(4-thiomorpholino)-pseudo-uridine triphosphate; 1-methyl-6-(substituted phenyl)pseudo-uridine triphosphate; 1 -methyl-6-amino- pseudo-uridine triphosphate; 1-methyl-6-azido-pseudo-uridine triphosphate; l-methyl-6- bromo-pseudo-uridine triphosphate; 1-methyl-6-buty 1-pseudo-uridine triphosphate; 1-methyl- 6-chloro-pseudo-uridine triphosphate; 1 -methyl-6-cyano-pseudo-uridine triphosphate; 1- methyl-6-dimethylamino-pseudo-uridine triphosphate; 1 -methyl-6-ethoxy-pseudo-uridine triphosphate; 1-methyl-6-ethylcarboxy late-pseudo-uridine triphosphate; 1 -methyl-e-ethyl- pseudo-uridine triphosphate; 1-methyl-6-fluoro-pseudo-uridine triphosphate; 1 -methyl-e- formyl-pseudo-uridine triphosphate; 1-methyl-6-hydroxy amino-pseudo-uridine triphosphate; 1-methyl-6-hydroxy-pseudo-uridine triphosphate; 1-methyl-6-iodo-pseudo-uridine triphosphate; 1-methyl-6-iso-propyl-pseudo-uridine triphosphate; 1-methyl-6-methoxy- pseudo-uridine triphosphate; 1-methyl-6-methylamino-pseudo-uridine triphosphate; 1 -methyl- e-phenyl-pseudo-uridine triphosphate; 1-methyl-6-propyl-pseudo-uridine triphosphate; 1- methyl-6-tert-butyl-pseudo-uridine triphosphate; 1 -methyl-6-trifluoromethoxy-pseudo-uridine triphosphate; 1-methyl-6-trifluoromethyl-pseudo-uridine triphosphate; 1- morpholinomethylpseudouridine triphosphate; 1 -pentyl-pseudo-uridine triphosphate; 1- phenyl-pseudo-uridine triphosphate; 1-pivaloylpseudouridine triphosphate; 1- propargylpseudouridine triphosphate; 1 -propyl-pseudo-uridine triphosphate; 1-propynyl- pseudouridine; 1-p-tolyl-pseudo-uridine triphosphate; 1-tert-Butyl-pseudo-uridine triphosphate; 1-thiomethoxymethylpseudouridine triphosphate; 1- thiomorpholinomethylpseudouridine triphosphate; 1 -trifluoroacetylpseudouridine triphosphate; 1-trifluoromethyl-pseudo-uridine triphosphate; 1 -vinylpseudouridine triphosphate; 2,2'-anhydro-uridine triphosphate; 2'-bromo-deoxyuridine triphosphate; 2'-F-5- methyl-2'-deoxy-uridine triphosphate; 2'-methoxy-5-methyl-uridine triphosphate; 2'-methoxy- pseudo-uridine triphosphate; 2'-a-ethynyluridine triphosphate; 2'-a-trifluoromethyluridine triphosphate; 2'-b-ethynyluridine triphosphate; 2'-b-trifluoromethyluridine triphosphate; 2'- deoxy-2',2'-difluorouridinetriphosphate; 2'-deoxy-2'-a-mercaptouridine triphosphate; 2'- deoxy-2'-a-thiomethoxyuridine triphosphate; 2'-deoxy-2'-b-aminouridine triphosphate; 2'- deoxy-2'-b-azidouridine triphosphate; 2'-deoxy-2'-b-bromouridine triphosphate; 2'-deoxy-2'-b- chlorouridine triphosphate; 2'-deoxy-2'-b-fluorouridine triphosphate; 2'-deoxy-2'-b- iodouridine triphosphate; 2'-deoxy-2'-b-mercaptouridine triphosphate; 2'-deoxy-2'-b- thiomethoxyuridine triphosphate; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O-methyl- 5-(l-propynyl)uridine triphosphate; 3-alkyl-pseudo-uridine triphosphate; 4'-azidouridine triphosphate; 4'-carbocyclic uridinetriphosphate; 4'-ethynyluridine triphosphate; 5-(l- propynyl)ara-uridine triphosphate; 5-(2-ruranyl)uridine triphosphate; 5-cyanouridine triphosphate; 5-dimethylaminouridine triphosphate; 5 '-homo-uridine triphosphate; 5-iodo-2'- fluoro-deoxyuridine triphosphate; 5-phenylethynyluridine triphosphate; 5-trideuteromethyl-6- deuterouridine triphosphate; 5-trifluoromethyl-uridine triphosphate; 5-vinylarauridine triphosphate; 6-(2,2,2-trifluoroethyl)-pseudo-uridine triphosphate; 6-(4-morpholino)-pseudo- uridine triphosphate; 6-(4-thiomorpholino)-pseudo-uridine triphosphate; 6-(substituted- phenyl)-pseudo-uridine triphosphate; 6-amino-pseudo-uridine triphosphate; 6-azido-pseudo- uridine triphosphate; 6-bromo-pseudo-uridine triphosphate; 6-butyl-pseudo-uridine triphosphate; 6-chloro-pseudo-uridine triphosphate; 6-cyano-pseudo-uridine triphosphate; 6- dimethylamino-pseudo-uridine triphosphate; 6-ethoxy-pseudo-uridine triphosphate; 6- ethylcarboxylate-pseudo-uridine triphosphate; 6-ethyl-pseudo-uridine triphosphate; 6-fluoro- pseudo-uridine triphosphate; 6-formyl-pseudo-uridine triphosphate; 6-hydroxy amino-pseudo- uridine triphosphate; 6-hydroxy-pseudo-uridine triphosphate; 6-iodo-pseudo-uridine triphosphate; 6-iso-propyl-pseudo-uridine triphosphate; 6-methoxy-pseudo-uridine triphosphate; 6-methylamino-pseudo-uridine triphosphate; 6-methyl-pseudo-uridine triphosphate; 6-phenyl-pseudo-uridine triphosphate; 6-propyl-pseudo-uridine triphosphate; 6- tert-butyl-pseudo-uridine triphosphate; 6-trifluoromethoxy-pseudo-uridine triphosphate; 6- trifluoromethyl-pseudo-uridine triphosphate; alpha-thio-pseudo-uridine triphosphate; pseudouridine l-(4-methylbenzenesulfonic acid) triphosphate; pseudouridine l-(4- methylbenzoic acid) triphosphate; pseudouridine triphosphate 1-[3-(2-ethoxy)]propionic acid; pseudouridine triphosphate 1-[3-{2-(2-[2-(2-ethoxy )-ethoxy] -ethoxy )-ethoxy}] propionic acid; pseudouridine triphosphate l-[3-{2-(2-[2-{2(2-ethoxy )-ethoxy} -ethoxy] -ethoxy )- ethoxy}] propionic acid; pseudouridine triphosphate 1-[3-{2-(2-[2-ethoxy ]-ethoxy)- ethoxy}] propionic acid; pseudouridine triphosphate 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; pseudouridine triphosphate 1-methylphosphonic acid; pseudouridine triphosphate 1- methylphosphonic acid diethyl ester; pseudo-uridine triphosphate-Nl-3-propionic acid; pseudo-uridine triphosphate-N1-4-butanoic acid; pseudo-uridine triphosphate-Nl-5-pentanoic acid; pseudo-uridine triphosphate-Nl-6-hexanoic acid; pseudo-uridine triphosphate-N1-7- heptanoic acid; pseudo-uridine triphosphate-Nl-methyl-p-benzoic acid; pseudo-uridine triphosphate-Nl-p-benzoic acid; wybutosine; hydroxy wybutosine; isowyosine; peroxywybutosine; undermodified hydroxywybutosine; 4-demethylwyosine; 2,6- (diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl: 1,3-( diaza)-2-( oxo )-phenthiazin- 1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2
(amino)purine; 2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido, 2'fluro-cytidine;2' methyl, 2'amino, 2'azido, 2'fluro-adenine;2'methyl, 2'amino, 2'azido, 2'fluro-uridine;2'-amino-2'- deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosinyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'- deoxyribose; 2'-fluoro-modified bases; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3- yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3-(methyl)-7- (propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4- (methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5 -nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6- (methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pynOlo-pyrimidin-2-on-3-yl; 7- (aminoalkylhydroxy)-l-(aza)-2-(thio )-3-(aza)-phenthiazin-1-yl; 7-(aminoalkylhydroxy)-l- (aza)-2-(thio)-3-(aza)-phenoxazin-11yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)- phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-( diaza)-2-( oxo )-phenthiazin-l-yl; 7- (aminoalkylhydroxy)-1,3-( diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7- (guanidiniumalkylhy droxy )- 1 -(aza)-2-(thio )-3 -(aza)-phenoxazinl-y 1; 7 -
(guanidiniumalkylhy droxy)- 1 -(aza)-2-(thio )-3-(aza)-phenthiazin-1-yl; 7 -
(guanidiniumalkylhy droxy )- 1 -(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -yl; 7-
(guanidiniumalkylhy droxy )-l,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(guanidiniumalkyl- hydroxy)-1,3-( diaza)-2-( oxo )-phenthiazin-1-yl; 7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-( oxo )-phenoxazin-1-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, 7-deaza- inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaza)-2- (oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl; aminoindolyl; anthracenyl; bis-ortho- (aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis-ortho-substituted-6-phenyl- pynOlo-pyrimidin-2-on-3-yl; difluorotolyl; hypoxanthine; imidizopyridinyl; inosinyl; isocarbostyrilyl; isoguanisine; N2-substituted purines; N6-methyl-2-amino-purine; N6- substituted purines; N-alkylated derivative; napthalenyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; 06-substituted purines; O-alkylated derivative; ortho-(aminoalkylhy droxy )-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl; oxoformycin triphosphate; para-(aminoalkylhydroxy)-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; pentacenyl; phenanthracenyl; phenyl; pyrenyl; pyridopyrimidin-3-yl; 2-oxo-7-amino- pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; pyrrolopyrimidinyl; pyrrolopyrizinyl; stilbenzyl; substituted 1,2,4-triazoles; tetracenyl; tubercidine; xanthine; xanthosine-5'- triphosphate; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyri din-d one ribonucleoside; 2-amino-riboside-triphosphate; formycin A triphosphate; formycin B triphosphate; pyrrolosine triphosphate; 2'-hydroxyl-ara-adenosine triphosphate; 2'-hydroxyl- ara-cytidine triphosphate; 2'-hydroxyl-ara-uridine triphosphate; 2'-hydroxyl-ara-guanosine triphosphate; 5-(2-carbomethoxyvinyl)uridine triphosphate; and N6-(19-amino- pentaoxanonadecyl)adenosine triphosphate.
[0062] Nucleosides described herein can also include modified sugars. 2'-Sugar modifications of the present disclosure include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O- alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula (O-alkyl)m, where m is 1 to about 10. Preferred among these poly ethers are linear and cyclic polyethylene glycols (PEGs), and PEG-containing groups, such as crown ethers and those which are disclosed by Ouchi et al, Drug Design and Discovery 1992, 9, 93; Ravasio et al. , J. Org. Chem. 1991, 56, 4329; and Delgardo et. al, Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249, each of which is hereby incorporated by reference in its entirety. Further sugar modifications are disclosed in Cook, Anti-Cancer Drug Design, 1991, 6, 585-607. Fluoro, O- alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitutions are described in U.S. Patent No. 6,166,197, entitled "Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2' and 5' Substitutions," hereby incorporated by reference in its entirety.
[0063] Additional 2'-sugar modifications of use in the present invention include 2'-SR and 2'- NR.2 groups, where each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2'-SR nucleosides are disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in its entirety.
[0064] The present invention also includes nucleic acids derivatized with selenium (Se). Examples of Se-derivatized nucleic acids include, but are not limited to, nucleic acids where O-atom at the positions 2', 4', and/or 5' of the sugar have been replaced with Se. Other examples include oxygen replacement with Se in nucleobases and non-bridging phosphates. Such nucleic acids are described in, for example, Pallan et al, Nat. Protoc., 2(3):647-51 (2007), and Nat. Protoc., 2(3);640-646 (2007), hereby incorporated by reference in their entirety.
[0065] Other examples of nucleic acids suitable for the present invention include boron containing nucleic acids, such as those described in Schinazi et al., Nucleosides and Nucelotides, 17(635-647 (1998); Biochem., 35(18):5741-5746 (1996); J. Org. Chem., 79(8):3465-3472 (2014), hereby incorporated by reference in their entirety.
[0066] Preferably, the methods disclosed herein further comprises washing the oligonucleotide-resin complex with an organic solvent after the immobilizing step. In other embodiments, the method also comprising drying the oligonucleotide-resin complex after the immobilizing step. The method may also comprise eluting the modified oligonucleotide from the resin by washing the oligonucleotide-resin complex with an aqueous buffer solution, once the modification of the DNA is complete.
[0067] Model DNA could readily undergo RASS (Strata XL-A resin, Phenomenex), but initial attempts to apply P(V)-conjugation proved difficult, furnishing only low yields of the modified product. On the basis of significant amounts of hydrolyzed P(V)-derivatives in the crude product mixture, it was postulated that this protocol (3x washes with dry acetonitrile) was not sufficient to fully dry the DNA-bound resin. Residual water would subsequently quench the P(V)-modules upon addition of DBU. Optimization of the washing protocol to use reagent- grade DMA, then THF, and finally drying under vacuum rectified this issue. With this revised protocol in hand, promising initial reactivity was observed (Fig. 3B, entry 1). By modulating DBU stoichiometry, concentration, and reaction time, general conditions were identified (Fig. 3B, entry 3) to produce singly labeled products (3) and (4) with good conversion (determined by total UV quantification at 260 nm) at both the 5' and 3' position, with no observable byproducts. The labeling position was confirmed by MS fragmentation. Also, DNA without any phosphate “blocking” groups could be selectively modified at the more reactive 5' terminus with a single P(V)-module by reducing P(V)-module concentration, albeit at reduced yields (53%). Additionally, the same DNA starting material (without terminal phosphates) could be dual labeled at both termini with two P(V)-modules under standard conditions (73%). DNA lacking a terminal phosphate could be readily phosphorylated by T4 polynucleotide kinase (T4PNK) (30 min) and directly loaded onto the support for subsequent SENDR protocol. Biochemically phosphorylated substrates were labeled at efficiencies comparable to chemically phosphorylated material. The conditions also proved to be sequence independent. SENDR provided efficient conjugations to oligonucleotides regardless of the identity of the terminal nucleoside. Also, both “sticky ends” (i.e., overhanging oligonucleotides) and “blunt ends” (i.e., nonoverhanging oligonucleotides) could be modified efficiently. This is important as there exists evidence that DNA retains its secondary structure while adsorbed to the support. High Tm DNA hairpins with nonoverhanging terminal alcohols were labeled in higher conversions when thermally denatured while being adsorbed to the support. With all of these elements combined, the development of SENDR was complete, and the inventor’s attention turned to its application. On the basis of previous studies, it was envisioned that a wide array of P(V)-modules derived from alcohol nucleophiles could be prepared as stable (and often crystalline) reagents. Combined with the present findings, subsequent coupling to DNA sequences would allow for a vast scope. Indeed, a large number of P(V)-modules (Fig. 2, ψ- 1-ψ-20), including multiple click chemistry handles, protected amines, an activated disulfide, an MRI probe, a fluorescent quencher, a ligand for radiomedicine, photoaffmity tag, a fluorophore, and nucleosides, were prepared. It is important to note that azide-containing handles typically cannot be incorporated directly in solid phase DNA synthesis by standard phosphoramidite chemistry as the P(III) containing phosphoramidite reactive group generally reduces the azide in a classical Staudinger reaction. Thus, most suppliers conjugate an azide to a preinstalled amine via NHS-ester chemistry postsynthesis. All P(V) ψ-modules produced singly labeled products upon conjugation to DNA in good to excellent conversion at both the 3' and 5' hydroxyl groups. DNA recovery was also good (30-80%), given that the upper yield limit after ethanol precipitation is known to be 80-85%. Throughout these applications, the general conditions were not modified, with the exception of cases where solubilization of the reagent required slightly elevated temperature (37 to 50 °C). The SENDR-modified oligonucleotides could be further processed via additional conjugation and click manipulations (Fig. 4). Thus, DNA-linked azides and alkynes were competent in SPAAC and CuAAC respectively, and directly provided constructs that were useful without further purification. In addition, DNA-linked azides could be easily transformed into the corresponding amines through the addition of a water-soluble phosphine (TCEP). This manipulation could be performed after SENDR as a one-pot procedure in the elution buffer, providing an exceedingly simple route to amine-modified DNA. Similarly, the construction of high-value DNA labeled with 3' TAMRA or biotin (Fig. 5). The 3' SENDR-modified DNA could be quantitatively transformed with multiple complex azides to furnish DNA conjugates that are sufficiently homogeneous (>80%) for most biochemical experiments without additional purification. Potential adverse effects on Cu(I) chemistry that could arise, due to coordination to the phosphorothioate moiety, were not observed, and CuAAC could be readily employed, allowing for, in principle, near-infinite diversification.
[0068] P(V) and PSI reagents disclosed herein can be readily derivatized with almost any alcohol allowing for almost unlimited scope in the production of P(V) modifications or handles. A large number of PSI or P(V) derived tags, which included multiple click chemistry handles, protected amines, an activated disulfide, MRI probe, fluorescent quencher, radio medicine ligand, photoaffinity tags, and nucleosides, were developed. All these reagents were found to produce singly labeled product in good to excellent yields at both the 3’ and 5’ hydroxyl group with good DNA recovery (50-80%) (Figure 3). Although the reaction conditions identified were readily implementable, some of these couplings proceeded better at 37°C, probably due to differences in solubility.
[0069] The PSI reagent molecule contemplated herein may comprise, but is not limited to, the following chemical formulas:
Figure imgf000029_0001
Figure imgf000030_0001
[0070] The PSI or P(V) reagents contemplated herein have a general formula
Figure imgf000030_0002
wherein X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’,
R’ is at each occurrence are independently selected from the group consisting hydrogen, C1to C10 alkyl, C1to C10 heteroalkyl, C1to C10 cycloalkyl, C1to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl Y and Z are each independently selected from the group consisting of O, S, and N
R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein: any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle.
[0071] The leaving group X reacts with a nucleophile to produce the PSI or P(V) module,
Figure imgf000031_0001
, which is then used for reacting with the nucleotide. Some non-limiting examples of P(V) reagents and their corresponding P(V) modules are shown below:
Figure imgf000032_0001
[0072] The labeled oligonucleotides could be directly manipulated with various readily utilized chemistries. DNA linked azides were competent in SPAAC and DNA-linked alkynes were competent in CuAAC directly providing constructs that were experimentally useful. DNA linked azides could be easily transformed into the corresponding amines through the addition of a water-soluble phosphine (TCEP). This approach to creating DNA linked amines presents cost saving opportunity, as SENDR derived modified oligo, for example 3’ DNA, labeled with TAMRA or Biotin would be more time and cost effective than the currently available modified oligonucleotides.
[0073] In one aspect, the SENDR technology has the advantage of being applicable to a variety of oligonucleotides. The inventors found that SENDR was able to efficiently modify both phosophothioate antisense oligonucleotides and large structured aptamers (Figure 4). In one embodiment, the 3’ phosphorylated version of Fomivirsen (Vitravene), which is an antisense antiviral drug used in the treatment of cytomegalovirus retinitis in immunocompromised patients, was efficiently modified with a number of reactive handles without modification of the basic protocol. Thus, the inventors contemplate creating pools of Antisense Oligonucleotides with handles for the attachment of various target engaging small molecules or peptides. In another embodiment, the inventors found that a large (58 nt) protein A aptamer which exhibits significant secondary could also be routinely modified in good conversion. This could be useful in the modification of entire SELEX pools with reactive warheads or target engaging moieties.
[0074] In some embodiments of the methods and techniques disclosed herein, the modified oligonucleotide is modified at the 5’ hydroxyl group. In other embodiments, wherein the modified oligonucleotide is modified at the 3’ hydroxyl group. In still other embodiments, the oligonucleotide is modified at both the 3’ end and the 5’ end. In such cases, the 5’ modification may be the same as the 3’ modification, or the 5’ modification may distinct and separate from the 3’ modification. Furthermore, in some embodiments, the oligonucleotide is a structured aptamer, a gene fragment, a primer, a hybridization probe, an oligonucleotide therapeutic, a protein recognition sequence, or a library of the aforementioned. The nucleotide may also be conjugated to a protein to form an oligonucleotide-protein conjugate. The method may further comprise incorporation of unnatural nucleotides into the nucleic acid sequence. Examples of unnatural nucleotides include polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides [0075] As discussed throughout this disclosure, the PSI linked small molecules comprises reactive handles, affinity tags, fluorophores, and FMRI probes, as described above. In some cases, the modified oligonucleotide may further conjugate with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins. The biorthogonal chemistry may comprise Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffmity labeling, and hydrazone ligation. In some embodiments, the method may further comprise chemical ligation of two strands of nucleic acids by reacting a first modified strand of the nucleic acid with another modified strand of nucleic acid using a biorthogonal reaction such as the CuAAC reaction.
[0076] The SENDR methods disclosed herein may also be used for diagnosing a disease in a patient in need thereof. The method comprises obtaining a tissue sample, blood sample, or body fluid swab from the patient; extracting oligonucleotides from the patient sample; labeling and/or modifying the oligonucleotide by the SENDR method disclosed herein; performing PCR reaction on the labeled and/or modified oligonucleotide; and analyzing the labeled and/or modified oligonucleotide to diagnose the disease. The disease may an infectious disease. The method of claim 50, wherein the disease is a bacterial or viral disease, and the patient is diagnosed with the disease if the analyzed labeled and/or modified oligonucleotide comprises the bacterial or viral DNA. In other embodiments, the disease may be a tumor, and the patient is diagnosed with the tumor if the analyzed labeled and/or modified oligonucleotide comprises cancer specific DNA mutations. The method may further comprise treating the patient with a known drug.
Reversible Adsorption of Solid Support (RASS)
[0077] The Reversible Adsorption of Solid Support (RASS) method of performing chemistry on-DNA was disclosed by the inventors in PCT /US2019/048570, which is incorporated by reference herein in its entirety, including the drawings
[0078] RASS makes use of non-covalent adsorption of DNA onto a solid support to facilitate solvent exchange from aqueous buffers, in which DNA is most soluble, into polar and nonpolar organic solvents that are otherwise considered as DNA incompatible. As used herein, the term “organic solvent” refers to any solvent except aqueous solutions. In some embodiments, the term “organic solvent” refers to any solvent containing carbon compounds. Examples of organic solvents include, but are not limited to, aromatic compounds, chloroform, alcohols, phenols, esters, ethers, ketones, amines, and nitrated and halogenated hydrocarbons.
[0079] Furthermore, the RASS strategy enables reactions to be performed in organic solvents at near anhydrous conditions. This, in turn, opens previously inaccessible chemical reactivities. The Reversible Adsorption to Solid Support (RASS) approach, as described further herein, enabled the rapid development of C(sp2)-C(sp3) decarboxylative cross-couplings with broad substrate scope, an electrochemical amination, and improved reductive animation conditions.
[0080] The RASS technique has several advantages over currently available methods. For example, a small molecule tethered to a DNA hairpin (headpiece domain) can be adsorbed to the solid support, transferred to organic solvent and undergo a range of chemical transformations in an organic solvent selected specifically for the chemical reaction, rather than to accommodate the encoding DNA. Another advantage of the approach is that excess reagents and solvent can be washed away leaving the small molecule-DNA conjugate adsorbed to the resin. This property allows for sequential reactions such as two-step transformations and deprotection reaction sequences to be performed in a facile manner. Following the desired chemical modifications, the small molecule- DNA conjugate can be released from the solid support for subsequent mixing and pooling steps. While many organic reactions can be performed in water, typically significant concessions are made with respect to reactant scope (low aqueous solubility), dilution, reaction time, yield and handling. The non-covalent adsorption technique disclosed herein would provide a solution to most of these problems, and significantly expand the tool kit of both reactants and reactions. Additionally, the DNA molecule itself is directly exposed to reactive species that can result in degradation of the encoding molecule. Adsorbing the DNA onto a solid support has significant potential to stabilize and shield duplex DNA, protecting it from unwanted chemical reactions.
[0081] In one embodiment, RASS includes a method of facilitating a chemical reaction in an organic solvent, comprising: providing a polynucleotide encoded chemical library, wherein the polynucleotide encoded chemical library members are immobilized to a solid support by non- covalent adsorption. The polynucleotide encoded chemical library member is immobilized by non-covalent adsorption of the polynucleotide moiety to the solid support. In one embodiment, the polynucleotide is a DNA, and the polynucleotide encoded chemical library is a DNA encoded chemical library. The feasibility of this approach has been demonstrated through screening a variety of chromatography resins for the required properties of adsorbing a chemically modified DNA hairpin, retention of the DNA during washing with organic solvents, and subsequent elution to obtain the intact DNA without significant handling losses or chemical modifications. In one embodiment, the solid support comprises an anion exchange resin. In one embodiment, the solid support comprises a cation exchange resin. In one embodiment, the solid support comprises SDB-L and/or C18 silica gel. In one embodiment, the solid support comprises a hydrophobic resin. In one embodiment, the hydrophobic resin comprises a reversed phase, hydrophobic interaction, strong cation, weak cation, and affinity chromatography. In one embodiment, the solid support comprises silica-based resin, crosslinked polystyrene, crosslinked glycan, crosslinked PEG, and combinations thereof.
[0082] This procedure identified a strong-anion exchange resin to which aqueous DNA can be adsorbed, transferred, and retained in a variety of organic solvents including: trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), 1,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), 1 ,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH). In one embodiment, the organic solvent comprises trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), 1,4-dioxane, teterahydrofuran (THF), dimethylacetamide (DMA), toluene, N-methylpyrrolidone (NMP), 1,2-dichloroethane (DCE), dimethylfomamide (DMF), and ethanol (EtOH).
[0083] While adsorbed, the inventors demonstrated a number of classic organic reactions such as amide coupling, benzimidazole formation, reductive animation, and Wittig, Henry and oxime reactions in solvents varying from DMA to DCM. Further, the compatibility of the electrostatically adsorbed DNA with palladium catalysis including Sonogashira cross-coupling reactions has been demonstrated, as disclosed herein. Importantly, all these reactions were demonstrated through a sequential series of an initial amide bond formation on the amino- linker- DNA hairpin, washing, solvent exchange, followed by a second reaction, all while adsorbed on the resin.
[0084] The immobilized polynucleotide as disclosed herein can undergo selective chemical modification while adsorbed to the solid support and exposed to organic solvent. In one embodiment, the chemical modification comprises an amide coupling in methylene chloride.
In one embodiment, the chemical modification comprises a Suzuki coupling in dimethylacetamide. In one embodiment, the chemical library members undergo single or multiple synthetic reaction steps while being immobilized to the solid support. In one embodiment, the synthetic reaction steps may be in the same or in different solvents. In one embodiment, the method further comprises washing away small molecules and/or reagents while leaving the polynucleotide encoded chemical library immobilized to the solid support.
[0085] In one embodiment, the RASS method may be used in challenging reactions such as decarboxylative cross couplings and CH activations. The chemical composition of the linker between the DNA and the encoded small molecule may also be used to enhance the performance of a specific reaction. While the current anion exchange resin is highly promising, the inventors have found the current method to be compatible with a wide range of resins (anion exchange and others). Furthermore, distinct resins should be used to facilitate specific desired chemical transformations. The technology would allow adaptation of previously incompatible reactants and chemistries and facilitate access to previously inaccessible chemical and conformational diversity.
[0086] In one embodiment, the RASS method further comprises a covalent tag that modulates binding to the solid support and removal from the solid support. In one embodiment, the method further comprises a linker between the polynucleotide and the chemical library member. In one embodiment, the linker modulates steric or conformational interactions between the site of reaction, polynucleotide, and the solid support. In one embodiment, the linker facilitates the desired chemical reaction. In one embodiment, the solid support is modified with ligands, catalysts etc. to affect the desired reaction.
[0087] The concept of adsorbing biological macromolecules onto solid support to facilitate chemical transformations has been developed in the context of peptide modification. The observation that the polyvalent adsorption kinetics of biomacromolecules compared to monovalent small molecules allows for their selective binding and release from various solid supports, distinct from their physical properties such as hydrophobicity. Recently, this phenomenon was exploited to perform selective chemical transformation of peptides and proteins while adsorbed to reversed phase silica in aqueous and organic systems. This Reversible Adsorption to Solid Support (RASS) approach has enabled solvents to be exchanged, excess reagents to be washed away, and sequential reactions to be performed on the adsorbed macromolecules without the need for intermediate purification and isolation steps.
[0088] Recent work by the inventors exploited peptide and protein immobilization as a tool for synthetic chemistry. The Reversible Adsorption to Solid Support (RASS) approach leverages the multivalent binding kinetics of biomacromolecules to selectively bind to and elute from an inert solid support, such as reversed phased silica. See Cistrone, et al, Click-Based Libraries of SFTI-1 Peptides: New Methods Using Reversed-Phase Silica. ACS Comb. Sci. 2016, 18, 139-143, which is incorporated by reference herein in its entirety. Critically, the differences in binding kinetics of biomacromolecules and small molecules allow for adsorbed biomolecules to react with small molecule reagents with the same logic as employed in conventional (covalently bound) solid phase organic synthesis strategies. See Flood, et al Post-Translational Backbone Engineering through Selenomethionine-Mediated Incorporation of Freidinger Lactams. Angew. Chem., Int. Ed. 2018, 57, 8697- 8701, which is incorporated by reference herein in its entirety. Thus, excess small molecule reagents are simply washed away while the polyvalent macromolecule remains adsorbed to the solid support. This allows for the use of organic solvents and reaction conditions that would be otherwise incompatible for the biomolecule.
[0089] A mixed mode polystyrene strong anion exchange resin, (for example, Phenomenex, Strata-XA) which contains a butyl quaternary ammonium moiety, was found to be an excellent platform for RASS on DNA (Figure 5B). Such resins incorporate both hydrophobic interactions (polystyrene, butyl substituents) and electrostatic interactions (quaternary amine) and effectively anchor the DNA in a polyvalent manner. When a model headpiece with a pendent free amine, in PBS (100 mM DNA), was added to the resin, it efficiently adsorbs to the surface, as indicated by the lack of DNA detected in the binding supernatant (Figure 5C). The bound DNA could then be eluted into a high salt elution buffer, an approach that is widely applied in molecular biology. Importantly, the Strata-XA resin allows the DNA- resin complex to be transferred from aqueous solution into neat organic solvents. Following removal of the solvent from the resin, the DNA can be washed with aqueous buffer, eluted from the resin with salt, and isolated through ethanol precipitation. Failures encountered with numerous other resins that were tested can be attributed to a lack of strong initial DNA binding or lack of retention in organic solvents. This represents a robust platform for the controlled, reversible binding and elution of DNA fragments to facilitate manipulation in organic media in ways that are not possible using conventional aqueous techniques. As an initial proof of concept, a simple amide bond formation reaction was performed in CH2C12. Alternative solvents such as THF, DMF, Dioxane, DMSO, and MeCN were also compatible, with observed yields mirroring solvent dependencies for small molecule carbodiimide couplings. SENDR: Complex Settings
[0090] SENDR could also be adapted for the efficient modification of phosphorothioate antisense oligonucleotides (ASOs) (Fig. 5). The 3' phosphorylated version of Vitravene, an FDA-approved ASO for the treatment of cytomegalovirus retinitis (CMV), was efficiently ligated with a number of P(V)-modules with no change to the general protocol. The 3' phosphorylated version of MALAT1, a published ASO with multiple modified sugars and bases, and containing PS linkages, could be readily modified with multiple P(V)-modules. This result exemplifies the opportunity for the late-stage modification of ASO pools with target- engaging small molecules or peptides. Previously, such handles would have required a de novo chemical synthesis for each new compound. A large (58 nt) “protein A” aptamer (54) which exhibits significant secondary structure could be modified at the 3' hydroxyl in 70% conversion (Fig. 6). Additionally, an aptamer to human neutrophil elastase (hNE) (56) could be modified at the 3' position with a phosphorothioate electrophile and was labeled using both enantiomers of (ψ-14), in similar conversions. The unlabeled aptamers were selectively digested using ExoIII leaving high purity aptamers (Sp-(57)) and (Rp-(57)). The addition of the phenol (Fig. 6) electrophile (Sp-(57)) and (Rp-(57)) conferred a 10-fold increase in potency (Ic50 of 15 and 17 nM respectively) when compared to parent aptamer (63) (Ic50 of 140 nM) in a fluorescence- based hNE inhibition assay. Also small molecules containing the same electrophiles were inactive in this screen. This example further demonstrates the sequence and structure independence of the SENDR platform. It could prove useful in the modification of entire SELEX pools with libraries of reactive warheads or target engaging moieties for the facile creation of DNA-small molecule chimeric inhibitors.
SENDR: DNA - Protein conjugates
[0091] Another utility of SENDR was also demonstrated in the formation of oligonucleotide- protein conjugates, which are becoming increasingly valuable in the production of long-acting and/or targeted oligonucleotide drugs. An oligonucleotide was modified by SENDR (using ψ- 8) with an activated disulfide group resulting in (61) (Fig. 8A). This construct could be used directly, without purification in a disulfide forming reaction with bovine serum albumin (BSA). The reaction cleanly furnished the DNA-BSA conjugate (62), and ESI-TOF analysis of the crude reaction mixture indicated that no unmodified BSA remained in solution. Conjugation to serum albumin is a valuable half-life increasing strategy for quickly cleared peptide and small protein drugs and could in principle be applied to next-generation ASOs. [0092] SENDR was also used to create a DNA construct that could be used in site-specific antibody conjugations (Fig. 8B). The complementary cDNA sequence of FDA-approved ASO Tegsedi (cTegsedi) was modified with an alkyne handle. In turn, this alkyne was ligated to a reactive beta-lactam containing moiety via CuAAC. The beta lactam containing oligonucleotide (64) was competent in the site-specific labeling of an engineered lysine on the heavy chain of a IgG that has in use in antibody drug conjugates (ADCs). This system was derived from the antihapten mAh h38C2 and is especially reactive toward beta lactam haptens. The reactive lysine residue also catalyzes a retro aldol reaction with methodol, which results in increased fluorescence of the aldehyde product. The modified site on the antibody was confirmed to be the catalytic lysine via methodol florescence assay — after conjugation, signal from the florescent aldehyde was not detected (Fig. 8B). These antibodies have shown promise as flexible platforms for the production of antibody drug conjugates, as they can be produced by typical recombinant methods, and drug molecules can be added at a known stoichiometry, to a known position, through a stable amide linkage. Labeling the antibody with cTegsedi created, in effect, an ASO delivery system that could protect, target, and deliver Tegsedi to the cell of interest. This process may be useful in the creation of many antibody-ASO conjugates that could provide targeted ASO therapies. The above two examples enabled by SENDR are striking due to the ease and efficiency with which these complex conjugates could be prepared, along with the near-infinite flexibility in design.
[0093] In another aspect, the SENDR technology as disclosed herein provides access to oligonucleotide-protein conjugates. SENDR enables formation of oligonucleotide protein conjugates, which are becoming increasingly valuable in the production of long acting and or targeted oligonucleotide drugs. First a DNA construct was derivatized with an activated disulfide group by SENDR. The resulting construct could be used directly, without purification in a disulfide forming reaction between the DNA construct and bovine serum albumin (BSA) cleanly yielded the DNA-BSA conjugate. ESI-ToF analysis of the crude reaction mixture indicated that no unmodified BSA remained in solution. Conjugation to serum is a half-life increasing strategy commonly employed in the context of readily cleared peptide and small protein drugs which could be applicable to ASOs.
[0094] SENDR was also used to create a DNA construct that could be readily used in site specific antibody conjugations. The complementary DNA sequence of approved ASO Tegsedi (cTegsedi) was modified with an alkyne handle to which a reactive beta-lactam containing moiety was clicked on. This beta lactam containing DNA was competent in the site specific labeling of an engineered lysine on the heavy chain of an IgGthat is especially reactive toward beta lactam heptans. This lysine can also catalyze a retro aldol reaction and will cause in increase in fluoresces of a fluorogenic aldehyde derived from methadol. The modified site on the antibody was confirmed to be the catalytic lysine by a methadol florescence assay in which signal from the florescent aldehyde was not detected. These antibodies have shown promise as flexible platforms for the production of antibody drug conjugates, as they can be produced by typical recombinant methods and drug molecules can be added at a known stoichiometry, to a known position. Labeling the antibody with cTegsedi created, in effect, an ASO delivery system that would protect, target and deliver Tegsedi to the cell of interest. This process would be useful in the creation of many antibody-ASO conjugates that could provide targeted ASO therapy.
[0095] Throughout the disclosure, it should be noted that while some of the embodiments and examples are described by using DNA construct or RNA construct, the same techniques could be used for any oligonucleotides the methods disclosed herein, and all such oligonucleotides are explicitly contemplated.
SENDR: Dual Labeled probes
[0096] Although many oligonucleotide-based technologies only require a single probe, the true power of hybridization probes is realized in dual labeled form. The canonical dual labeled oligonucleotide probe has a fluorophore label on one terminus and a fluorescence quencher at the other (Fig. 9A). Fluorescence of the probe is quenched when the two components are in close proximity. Molecular beacons (MBs), for example, form a stem loop system that brings the termini labels into close proximity when the target is not present (Fig. 1A). Upon target engagement, the MB adopts an extended conformation, which moves the two labels out of
FRET range and results in a fluorescence signal. Another ubiquitous example of dual labeled probes is the TaqMan qPCR probe. These probes are also typically constructed with a fluorophore and a quencher at the termini. These probes hybridize to a diagnostic sequence of interest and upon PCR elongation by Taq polymerase, the probe is cleaved (by the intrinsic exonuclease activity of Taq), and increased fluorescence is read out (Fig. 1A). Although
TaqMan PCR is widely considered to be the state-of-the-art in real-time PCR methods, practitioners are reliant on vendors for custom synthesis of probes with proprietary linking technologies. This synthesis must be done for each individual target and can prove to be prohibitively expensive. Indeed, the less sensitive method of SYBR Green-based qPCR, which relies on increased fluorescence of an intercalating dye during polymerization, is gaining in popularity because of the immense cost of buying custom TaqMan PCR probes for every experiment. SENDR, when used in concert with ubiquitous biochemical techniques, presents a unique opportunity for biochemical researchers to produce dual labeled probes for their own custom applications (Fig. 9A). Synthesis of these probes proceeds through a multistage process. In the event, a typical synthetic oligonucleotide containing a 5' phosphate and a 3' hydroxyl group is ligated with an alkyne group by SENDR (Fig. 9). Next, this modified oligomer is quantitatively dephosphorylated by recombinant shrimp alkaline phosphatase (rSAP) unmasking the 5' hydroxyl group, while the unmodified DNA is selectively degraded by ExoIII in one pot. This oligomer is then subjected to a second SENDR modification at the 5' terminus, providing the dual labeled probe. Similarly, remaining unlabeled oligonucleotide is selectively degraded by a mixture of lambda exonuclease and T4PNKin one pot. The T4PNK phosphorylates the remaining unlabeled 5' hydroxyl oligonucleotide which is then recognized and degraded by lambda exonuclease. These resulting probes are highly pure without the need for HPLC purification. The position of each label on the dual labeled probe was confirmed by MS fragmentation. This dual-labeled parent probe (68) is now primed for subsequent SPAAC/CuAAC reactions with any fluorophore/quencher pair desired to furnish the qPCR- competent probe. These reactions with FAM-DBCO and BHQ1 -azide proceed quantitatively, resulting in the qPCR competent construct (69). The utility of in-house probe production was demonstrated by the facile and expedient production of dual labeled probes for COVID-19 diagnostics (Fig. 9B). The native sequences for the panel of RT-PCR probes for COVID-19 diagnostics 66 and 67 were transformed in parallel (~48 h) into dual labeled probes through the above sequences in good overall conversions. Although this class of probes are commercially available, we have demonstrated an alternate paradigm for their synthesis, and we believe this could enable the construction of probes beyond those that are currently offered by vendors.
[0097] In yet another aspect, the SENDR technology was found to be useful for synthesis of dual labeled probes. Although many oligonucleotide based technologies only require a single probe, the true power of a oligonucleotide probe is realized in the dual labeled form. The canonical dual labeled probe has a fluorophore label on one terminus and a fluorescence quencher at the other. Fluorescence of the probe is quenched when the two components are in proximity. Molecular bacons, for example, form a stem loop system which brings the termini labels are in proximity when the target is not present. Upon target engagement, the MB adopts an extended confirmation taking the two labels out of FRET range and resulting in a fluorescence signal. Another ubiquitous example of these dual labeled probes is the TaqMan PCR probes. These probes are also typically constructed a fluorophore and a quencher at the termini. These probes hybridize to the diagnostic sequence of interest and upon PCR elongation by Taq polymerase the probe is cleaved (by the intrinsic exonuclease activity of Taq) and increased fluorescence is read out. Although TaqMan PCR is widely used, the requisite dual labeled probes have to be custom synthesized for each target of interest, which in turn can prove to be expensive.
[0098] The SENDR technology, as disclosed herein, when used in concert with ubiquitous biochemical techniques presents a unique opportunity for the typical biochemical researcher to cheaply produce dual labeled probes for their own custom applications. A typical synthetic oligonucleotide containing a 5’ phosphate (the modification that would result from biochemical production) and a 3’ hydroxyl group was modified with an azide group by SENDR. This modified oligomer was then quantitatively dephosphorylated with recombinant Shrimp Alkaline Phosphatase (rSAP) to “deprotect” the 5’ hydroxyl group. This oligomer is now competent for a second modification at the 3’ end providing the dual probe in 65% crude conversion. The position of each label on the dual labeled probe was confirmed by MS fragmentation. This dual labeled probe may be further modified through a SPAAC and a subsequent CuAAC reaction to furnish a qPCR competent probe.
[0099] It should be appreciated that the methods disclosed herein may be used to make combinatorial libraries and/or pools of oligonucleotides and primes for NGS, FACS, or other high throughput applications.
SENDR for COVID diagnostics
[00100] In yet another aspect of this disclosure, the SENDR method may be used for diagnosing a disease. The disease may be an infectious disease, such as a bacterial infection or viral infection.
[00101] In one embodiment, the SENDR protocol for diagnosing a viral disease, a coronavirus infection such as COVID-19, is described herein. SENDR method was used to make dual ladled probes for COVID-19 diagnostics. The native sequences for the panel of RT- PCR probes for COVID diagnostics, as defined by HHS 24 Jan 2020, were transformed in parallel (~48 hrs) into dual labeled probed through the above method in good overall yield. In times of pandemic, such as COVID- 19, when the commercial capacity for diagnostic probe creation is being shuttled towards the clinic, SENDR could provide health professionals and researchers with a time efficient probe source allowing continued therapeutic and vaccine research. In one embodiment, an exemplary compounds useful as a probe for coronavirus qPCR comprise the following, wherein N3 is an oligonucleotide from a coronavirus. The corresponding sequence listing is SEQ ID NO: 1.
Figure imgf000044_0001
[00102] For diagnosing whether a patient has a coronavirus infection, it is contemplated that a swab is taken from the nostril or throat of the patient. RNA present in the swab is extracted, and analyzed using the labeled PCR probe as disclosed throughout the disclosure. A PCR reaction is then performed to determine if coronavirus RNA is present in the patient sample. In one embodiment, the coronavirus disease is COVID-19.
SENDR kit
[00103] In another aspect of this disclosure, the inventors contemplate the use of SENDR reagents in a kit format. The process is simple and robust to be miniaturized and performed in a cartridge/flow set up and all reagents employed are shelf stable indefinitely. The inventors found that reaction set up when performed in a cartridge proved simpler and faster than the typically employed microcentrifuge tubes. Also, reaction efficiency was identical to reactions performed in microcentrifuge tubes. Thus, by using this kit a researcher could modify synthetic or biochemically derived oligonucleotide [00104] In this aspect of the instant disclosure, a kit is provided for oligonucleotide modification. The kit comprises one or more P(V) reagents as discussed throughout this disclosure; 1,8-Diazabicyclo[5.4. 0]undec-7-ene (DBU); Dry Tetrahydrofuran (THF); Dimethylacetamide (DMA); Elution buffer; and Strata AXL resin in fitted cartridge. Preferably, the elution buffer comprises 1M NaCIO4, 20% MeOH, 40 mM Tris pH 8.5.
[00105] From a pragmatic standpoint, a simple kit-format would be of use to the community. Toward that end, a “SENDR kit” was created from readily available consumables. The SENDR process is simple and robust enough to be miniaturized and performed in a cartridge/flow set up, and all reagents employed are shelf-stable indefinitely. Gratifyingly, when performed in a cartridge, the process proved simpler and faster than the previously employed microcentrifuge tubes. Importantly, reaction efficiency was identical to reactions performed in microcentrifuge tubes. Using this kit, a researcher could customize synthetic or biochemically derived oligonucleotide, in-house, with a suite of commercialized reagents. We believe that SENDR kits will expand the toolbox and allow researchers to pursue experimental designs that were previously out of reach.
[00106] As disclosed throughout, SENDR has the ability to easily make site-specific oligonucleotide modifications. Advantageously, SENDR it does not require de novo synthesis (and specialized equipment) and benefits from the intrinsic advantages of a late-stage incorporation. With the acknowledgment that enzymatic means of site-selective functionalization are powerful, SENDR is uniquely versatile and programmable using easily accessible reagents. As more modules are developed, one could imagine an unlimited diversity being incorporated. Regarding limitations, highly lipophilic groups are challenging to employ, groups sensitive to DBU might be problematic, and at this point are limited to terminal modifications. More broadly, the modular nature of the process could permit a more medicinal chemistry mindset into the derivatization of complex oligonucleotide-based conjugates. Although some may interject that SENDR-based modification of oligonucleotide lies outside the skill set of the general molecular biology practitioner, the robust chemistry should prove simple enough for any practitioner with basic liquid handling skills. The present disclosure demonstrates conjugations that are compatible with simple organic molecules, proteins, aptamers, and ASOs. Numerous extensions such as applications to carbohydrate conjugation, and multiplexed high throughput array is possible and contemplated herein.
SENDR for targeted delivery and/or improving half-life of therapeutic oligonucleotides [00107] In contrast to small molecule drugs and biologies which target gene products (i.e. proteins), nucleic acid therapeutics have the potential to therapeutically regulate essentially any gene of interest at the DNA or RNA level. Their versatility in treating inherited or acquired disorders stems from the ability to induce efficient gene silencing (inhibiting pathological/mutant protein production), gene expression (producing therapeutic proteins) or gene editing (correcting dysfunctional/mutated genes). Nucleic acid therapeutics or oligonucleotide therapeutics include antisense oligonucleotides (ASO), small interfering RNA (siRNA), plasmid DNA (pDNA), messenger RNA (mRNA), and complexes containing guide RNA (gRNA) as part of gene editing approaches.
[00108] Despite these advantages, using oligonucleotide as a therapeutic in vivo is challenging because of their unfavorable physicochemical characteristics, such as negative charge and relatively large size, which prevents their efficient uptake into cells. Furthermore, oligonucleotides are susceptible to degradation by nucleases in the circulation, suffer from rapid renal clearance, and induce immunostimulatory effects via pattern recognition receptors, resulting in adverse effects. Furthermore, these oligonucleotide therapeutics are potentially immunogenic and may require a delivery vehicle for efficient and specific transport to target cells and across the lipid bilayer. The SENDR method of oligonucleotide modification as presented herein is able to overcome a number of these disadvantages inherently present in the currently known oligonucleotide therapeutics.
[00109] Lipids and sugars are commonly used for targeted delivery of oligonucleotide therapeutic to a specific location in the patient body. For example, lipid nano particle (LNP) is used for directing an oligonucleotide therapeutic to the liver. Similarly, the modification at the 2'-position of the furanose sugar have been especially useful for improving the drug-like properties of antisense oligonucleotides (ASOs). The SENDR method as disclosed herein may be used to couple a sugar or lipid molecule to the oligonucleotide therapeutic to thereby target the delivery of the therapeutic oligonucleotide to the specific diseased cell location in the patient body. Viewed from a different perspective, the instant disclosure provides a method of targeting delivery of an oligonucleotide therapeutic to a specific location in the patient body, comprising: attaching a sugar or lipid to a therapeutic oligonucleotide by the SENDR method to thereby produce a modified therapeutic oligonucleotide; and administering the modified therapeutic oligonucleotide to the patient for targeted delivery to a specific location in the patient body. [00110] DNA and RNA-based therapeutics are also inherently unstable and prone to degradation by active and abundant nucleases (DNases and RNases). This hurdle of a short half life may be overcome by chemically modifying DNA or RNA to enhance its stability, and by employing synthetic carriers such as lipid nanoparticle (LNP) or polymer-based nanoparticle (PNP) systems for drug delivery.
[00111] The SENDR method as disclosed herein may be used for modifying the therapeutic oligonucleotides. Thus, in one embodiment, the present disclosure provides a method of increasing the half-life of an oligonucleotide therapeutic in-vivo, the method comprising: immobilizing the oligonucleotide on an inert resin to form oligonucleotide-resin complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-resin complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the inert resin, wherein the site specific P(V) reagent modification increases the half life of the oligonucleotide therapeutic in vivo.
[00112] The modified oligonucleotides prepared by the method above is contemplated to have stabilizing moieties such as modified nucleosides including pseudouridine, 5’-methyl- cytidine triphosphate (m5CTP), N6-methyl-adenosine-5’- triphosphate (m6ATP), 2-thio- uridine triphosphate (s2UTP), N6-methyladenosine (m6A), and N6,2-Odimethyladenosine (m6Am), a 5 ’cap, optimized 3’ poly (A) tail etc.
[00113] The modified oligonucleotides as prepared by the SENDR method would enhance the stability of the oligonucleotide drug, provide protection from nuclease degradation, confer drug-like properties to DNA and RNA, reduce immune stimulation, maximize on-target potency and prolong the duration of the drug.
[00114] The presently disclosed methods are further described in Flood et al, “Synthetic Elaboration of Native DNA by RASS (SENDR)” ACS Cent. Sci. 2020, 6, 1789-1799, which is incorporated by reference herein in its entirety, including supplemental information.
[00115] Embodiments of the present disclosure are further described in the following examples. The examples are merely illustrative and do not in any way limit the scope of the invention as claimed.
Example - Materials and methods [00116] Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), and dichloromethane (DCM) and acetonitrile (MeCN) were obtained by passing the previously degassed solvents through an activated alumina column. DMA was purchased from Sigma-Aldrich and used without further drying. P(V) reagents were purchased from Sigma Aldrich or synthesized as described below. DIC (N,N’-diisopropylcarbodimide) was purchased from Oakwood. Deionized water was used in all the reactions, unless otherwise stated. All the other reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. NaC104 was purchased at the highest commercial grade from Acros Organics. DNA tags was obtained from IDT, Inc., Coralville, IA. Recombinant All enzymes (Shrimp Alkaline Phosphatase (rSAP), ExoIII, T4PNK, and Lambda Exo) was obtained from New England Biolabs, Ipswich, MA. The Cut Smart buffer stock used enzymatic reactions was obtained from New England Biolabs, Ipswich, MA. UltraPureTM Agarose was obtained from Invitrogen, Carlsbad, CA. 50X TAE Buffer (Tris-acetate-EDTA) was obtained from Thermo Fisher Scientific, Waltham, MA. SYBRTM Safe DNA Gel Stain (10,000X) was obtained from Invitrogen, Carlsbad, CA. Gel Loading Dye, Purple (6X), no SDS was obtained from New England Biolabs, Ipswich, MA.
[00117] Yields under normal organic conditions refer to chromatographically and spectroscopically (1H, 31P NMR) homogeneous material, unless otherwise stated. TLC was performed using 0.25 mm E. Merck silica plates (60F-254), using short-wave UV light as the visualizing agent, and phosphomolybdic acid or KMn04 and heat as developing agents. NMR spectra were recorded on Bruker DRX-600, DRX-500, and AMX-400 instruments and are calibrated using residual undeuterated solvent (CHC13 at 7.26 ppm 1H NMR, 77.16 ppm 13C NMR). The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Column chromatography was performed using E. Merck silica gel (60, particle size 0.043-0.063 mm), and preparative TLC was performed on Merck silica plates (60F-254). High-resolution mass spectra (HRMS) were recorded on Agilent LC/MSD TOF mass spectrometer by electrospray ionization time of flight reflectron experiments. Melting points were recorded on a Fisher-Johns 12-144 melting point apparatus and are uncorrected.
[00118] PBS: Phosphate buffered saline was purchased from commercial and filtered before use. PBS is comprised of NaCl (137 mM), KC1 (2.7 mM), Na2HP04 (10 mM), KH2P04 (1.8 mM), at pH 7.4. [00119] Elute Buffer: DNA was eluted from resin using a sodium perchlorate buffer (1 M NaC104, 40 mM Tris, 20% MeOH, at pH 8.5). This was prepared from NaC104 (Acros Organics), and filtered after preparation. This buffer could be stored on the bench indefinitely.
[00120] HPLC-MS Analysis: Six microliter of the DNA solution was analyzed on a Waters H-Class LC with a Waters BEH Cl 8 column (2.1x55 mm, 1.7 pm, 130 A) using a gradient of 114 mM HFIP and 14 mM Et3N in water (A) and methanol (B) (0.3 mL/min, 10-26 %B over 5 minutes) at 60° C. Peak identities were determined by ESI- using the 6- ion.
[00121] HPLC-TOF High Resolution Analysis: One microliter of the DNA solution was analyzed on a Waters I-Class LC with a Waters BEH C18 column (2.1x55 mm, 1.7 pm, 130 A) using a gradient of 114 mM HFIP and 14 mM Et3N in water (A) and methanol (B) (0.3 mL/min, 10-26 %B over 10 minutes) at 60° C. Peak identities were determined by ESI- using the 3- ion.
[00122] Deconvolution: data visualization and integration were performed with Mass Lynx V4.1 software.
[00123] Conversion determination: the yields of on-DNA products were determined from UV absorbance trace (260 nm) peak area using the equation: Yield%= UV(prod)/UV (total DNA recovered), while ignoring UV coefficient difference for all DNA products and assuming 100% of DNA total recovery. While determining yields, any non-oligo material (as determined by close examination of mass spectra) that absorbed UV 260 nm was subtracted out of the final yield calculation. These peaks were determined to not contain oligo material when a DNA indicative mass spectra was not observed, and are usually attributed to small molecules not removed during the ethanol precipitation. These peaks were generally found at or before 1 minute retention time during analysis.
[00124] Preparative HPLC was performed on the Waters H Class instrument described above using customized gradients for each compound of interest and an automatic divert valve.
Example - Synthesis and Characterization of ψ-modules
[00125] The ψ-modules were prepared as follows: Alcohol (1.0 equiv.) and (-)-ψ (1.3 equiv.) were dissolved in anhydrous MeCN or DCM (0.1 M) in a flame-dried round-bottom flask. DBU (1.2 equiv.) was added to the reaction mixture while stirring. After 5-10 minutes, the crude reaction mixture was diluted with EtOAc or Et20 and transferred to a separatory funnel. The organic layer was washed with H20, saturated aqueous KH2P04 and brine. After drying over MgS04 and filtration, the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography unless otherwise stated
Example - SENDR Protocol, General Procedure 1A- Synthesis of ψ-modules
[00126] Nucleophile (1.0 equiv.) and P(V) reagent (1.3 equiv.) were dissolved in anhydrous MeCN or DCM (0.1 M) in a flame-dried round-bottom flask. DBU (1.2 equiv.) was added to the reaction mixture while stirring. After 5-10 minutes, the crude reaction mixture was diluted with EtOAc or Et2O and transferred to a separatory funnel. The organic layer was washed with H2O, saturated aqueous KH2PO4 and brine. After drying over MgS04 and filtration, the solvent was removed in vacuo. The product was purified by crystallization, silica gel column chromatography or used directly for the next step if in sufficient purity.
Example - SENDR Protocol, General Procedure IB: In Situ Module Formation
[00127] In some instances, it may be advantageous to directly prepare a module in solution and immediately use it in the SENDR process without isolation. To a solution of nucleophile (1.0 equiv.), chloroacetamide (1.0 equiv.) and P(V) reagent (1.3 equiv.) in anhydrous MeCN in a flame-dried round-bottom flask was added DBU (1.3 equiv.). After mixing for 5 minutes, the reaction was directly used in the SENDR protocol via Procedure 2 or 3.
Example - SENDR Protocol, General Procedure 2: SENDR in Microcentrifuge Tubes
Resin Preparation and DNA Loading
1. Cut the tip off of a 1 mL pipette tip and transfer resin (100 μL resin equilibrate in 1:1 PBS:MeCN, ~25 mg dry resin) with an adjustable volume pipette — Phenomenex Strata-XAL — into a 2 mL microcentrifuge tube.
2. Wash Resin with 500-1000 μL PBS. a. Spin down the resin slurry and aspirate the resin bed (by sucking off the supernatant with a glass pipet).
3. Add DNA (up to 50 nmol) in 100-500 μL PBS, vortex and incubate at room temp for 5-15 min.
Resin Washing and Drying
4. Wash loaded resin twice with DMA (500 μL)
5. Wash loaded resin three times with dry THF (500 μL) 6. Dry the resin for >2h in a speed vac or stuff a ball of paper in the top (so no resin can escape) and dry under vacuum for >2 h.
7. Resin Washing and Drying
Running the Reaction
8. Dissolve ψ-Module in dry MeCN (150 mM, 250 mM), and add it to the dry resin tube.
9. Add DBU (450 mM total, 18 μL) to the reaction tube.
10. Vortex for 30 seconds and incubate for 60 minutes at room temperature or 37 °C.
Working up Reaction
11. Aspirate reaction mixture and discard.
12. Wash reaction with MeCN or DMA (500 μL)
13. Wash with PBS or 1:1 PBS:MeCN (500 μL)
Elute DNA
14. Add 300 uL Elute Buffer (1 M NaClO4, 20% MeOH, 40 mM tris, pH 8.5) to resin bed.
15. Vortex for 30 seconds and agitate for 5-10 minutes.
16. Carefully collect the elution butter with micropipette without sucking up any resin.
17. Desalt with your preferred method.
[00128] In the above protocol, it should be noted that Strata-XAL if designed for large analytes. Strata-XAL is a 100 pm particle a with 300 A pore size. Moreover, “Washing the resin” refers to adding 500 to 1000 μL of solvent to the resin, vortexing (30 sec), spinning the resin down, and removing the supernatant with a pipette. Dry acetonitrile should be used for best reaction conversion. The elute buffer which contains 1 M NaClO4, 20% MeOH, 40 mM tris, pH 8.5 should be accessed with high quality NaClO.
Example 4 - SENDR Protocol, General Procedure 3: SENDR in Fritted Cartridges (Kit Format)
Preparation
1. Break off bottom of preloaded (with 100 μL Strata-XAL resin in 1:1 PBS:MeCN) Biorad column. Or transfer equilibrated resin (100 μL) to the column as above.
2. Allow packing solution to flow through into waste.
3. Add PBS (500 μL) and allow to flow through into waste.
DNA Loading:
4. Cap the bottom spout and add DNA (up to 50 nmol in 100-500 μL PBS) to column. 5. Cap the top, vortex and agitate for 5-15 min.
Resin Washing and Drying
6. Wash loaded resin twice with DMA (500 μL) and allow to flow to waste.
7. Wash loaded resin three times with dry THF (500 μL) and allow to flow to waste.
8. Replace the top cap but not the bottom and dry the resin for >2h in under vacuum. Running the Reaction
9. Add a NEW, CLEAN, and DRY cap to the bottom of the column.
10. Dissolve ψ-Module in dry MeCN (300 mM, 125 μL) or add the supplied PSI-Module solution to dry resin tube.
11. Add DBU in dry MeCN (300 mM, 125 μL) or add the supplied DBU solution to resin tube.
12. Replace both caps, vortex for 30 seconds, and incubate for 60 minutes at 37 °C. Working up Reaction
13. Remove both caps and allow the reaction mixture to drain to waste.
14. Wash reaction with MeCN or DMA (500 μL) allow to drain to waste
15. Wash with PBS or 1:1 PBS:MeCN (500 μL) allow to drain to waste Elute DNA
16. Replace bottom cap. Add 300 uL Elute Buffer (1 M NaClO4, 20% MeOH, 40 mM tris, pH 8.5) to resin bed and then replace top cap.
17. Vortex for 30 seconds and agitate for 5-10minutes.
18. Remove both caps and allow elute buffer to drain into a collection tube.
19. Desalt with your preferred method.
In the above protocol, it should be noted that flow throughs should be allowed to occur under gravity not under vacuum. Agitating the resin can be done with an end over end tumbler or an orbital shaker. Drying the resin under vacuum can be performed using a lyophilizer, high vac, or speed vac (if the columns fit).
Using the SENDR protocols described in the above examples, the inventors prepared a variety of modified oligonucleotides. Some exemplary reactions are shown below. In each of these examples, N refers to any unspecified nucleotide, and may be guanine or adenine or thymine or cytosine or uracil, or any modifications thereof:
1. Reaction Scheme A
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Example - Optimization of SENDR protocol and conditions
[00129] Optimization Procedure: SENDR was optimized by performing reactions in a microcentrifuge tube as described in General Procedure 2 with various reaction conditions (as described in the main text and Figure 2B). Briefly, 5 nmol of DNA was loaded onto 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. In the case in which stringent drying was not performed, the loaded resin was simply washed with acetonitrile (500 μL three times).
[00130] P(V) reagent in MeCN (varying concentration, 250 μL) was added to the loaded and dried resin, Then DBU (various concentration) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at room temperature for various amounts of time. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5- 10 minutes. The DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (-1000 μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the supernatant ethanol was decanted off. The tubes were dried via speed vacuum, the DNA was dissolved, and HPLC-MS analysis was performed.
[00131] SENDR was also performed on High Tm Hairpin. A hairpin oligo was incubated for 10 minutes at either RT or 66°C in 100 μL PBS. This solution was pipetted directly onto washed support (50 μL) and quickly vortexed. The reactions were allowed to cool to room temperature before washing and drying was performed according to general procedure 2. SENDR derivatization was performed under standard conditions.
Example - SENDR protocol for various alcohol selective reactions
[00132] The SENDR method as disclosed herein may be used for various alcohol selective reactions. The following alcohol selective reactions are described; however it should be recognized that the present disclosure is not limited to the reagents or reactions below, and these are merely presented as examples.
Figure imgf000059_0001
[00133] DNA 2 was dissolved (90 mM) in MES buffer (50 mM) at pH 6.2 containing phenethylaime (5 mM) and imidazole (20 mM). to this solution was added l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) (10 μL) from a stock solution in DMA (50 mM). This reaction was quickly vortexed and allowed to incubate at room temperature for 60 minutes. After 60 minutes the DNA was isolated by ethanol precipitation (previously described) and analyzed by liquid chromatography-mass spectrometry (LCMS).
Figure imgf000059_0002
[00134] DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. Phorphoramitide (150 mM) and tetrazole (450 mM) were added in dry MeCN and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resi was washed once with dry MeCN (500 μL) and oxidation solution was added (iodine, pyridine, water in THF). This was reaction incubated for 15 minutes and then the resin bed washed with MeCN and PBS. The DNA was eluted and precipitated as previously described and finally analyzed by LCMS.
Figure imgf000060_0001
[00135] DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. Tosyl Chloride (150 mM) and Collinide (450 mM) were added in dry MeCN and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resin was washed once with dry MeCN (500 μL). The DNA was eluted and precipitated as previously described and finally analyzed by LCMS.
Figure imgf000060_0002
[00136] DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. Nirtopheol (150 mM) and Triphenylphosphine (150 mM) were added in dry MeCN. DIAD (150 mM) was then spiked in and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resin was washed once with dry MeCN (500 μL). The DNA was eluted and precipitated as previously described and finally analyzed by LCMS. Low DNA recovery was observed by UV Vis absorbance (nanodrop).
Figure imgf000060_0003
[00137] DNA 1 was loaded onto the support according to General Procedure 2 and dried accordingly. DBU (450 mM) was added in dry MeCN and this was added to the resin from 5 minutes after 5 minutes benzyl bromide (150 mM) was then spiked in and the reaction was allowed to incubate at room temperature for 60 minutes. After 60 minutes the resin was washed once with dry MeCN (500 μL). The DNA was eluted and precipitated as previously described and finally analyzed by LCMS.
Example - Labeling of DNA Without Phosphate Blocking Groups
[00138] General Protocol and Optimization: SENDR on non-phosphate DNA was optimized by performing reactions in a microcentrifuge tube as described in General Procedure 2 with various reaction conditions (Figure 3B). Briefly, 5 nmol of DNA was loaded onto 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. In the case in which stringent drying was not performed (Figure 3B, Entry 8) the loaded resin was simply washed with acetonitrile (500 μL three times).
[00139] ψ-1 in MeCN (varying concentration, 250 μL) was added to the loaded and dried resin. Then DBU (various concentration) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at room temperature for various amounts of time. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the supernatant ethanol was decanted off. The tubes were dried via speed vacuum, the DNA was dissolved and HPLC-MS analysis was performed.
[00140] Deconvoluted Mass Spectrum: A crude solution of the reaction mixture in water (100 μM) was injected on the Waters I-Class ToF for peak identification. The MS cone voltage was then increased from 5 mV to 30 mV and the same sample was reinjected to fragment the modified oligonucleotide. Diagnostic mass fragments were then extracted, and their presence confirmed in the total ion current.
Example - T4PNK Phopshorylation then SENDR
[00141] Nonphosphorylated DNA was phosphorylated at the 5’ end by T4PNK. Briefly, 5 nMol DNA was added to a PCR tube and contiaining CutSmart (IX), 1 mM ATP, and 5 mM DTT (from 5x stocks). T4PNK was added (2 uL) and the reaction volume was brought to 50 μL total. The reaction tube was allowed to incubate for 30 minutes at 37°C. After 30 minutes the reaction mixture was added directly to the Strata XL-A support in PBS (500 uL) and vortexed. The support was dried according to General Procedure 2, and standard SENDR conditions were applied.
Example - Sequence Independence
[00142] SENDR was optimized by performing reactions in microcentrifuge tube as described in General Procedure 2. Briefly, 5 nmol of DNA was loaded onto 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum.
[00143] ψ-1 in MeCN (150 mM, 250 μL) was added to the loaded and dried resin. Then
DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at room temperature for various amounts of time. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolated via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of aNaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed. The method above was performed on a variety of substrates and a variety of DNA. Some of the modified oligonucleotides obtained by this method are illustrated below in table 1, and the corresponding sequence listings are SEQ ID NOs:2-5. Table 1.
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Example - SPAAC Reaction:
Figure imgf000070_0001
[00144] Compound 19 (5 nmol) was synthesized by General Procedure 2 and isolated crude after ethanol precipitation. Solid 19 was dissolved (98 μL) in tris buffer (50 mM) at pH 8.5. To this solution BDP-FL (2 μL) was added from stock solution (5 mM) in DMA, to a final concentration of 100 μM. This solution was vortexed and transferred to an HPLC vial. This reaction was injected onto the H-Class HPLC after 60 minutes. SPAAC product 43 was characterized below.
Example - CuAAC Reaction:
Figure imgf000070_0002
[00145] Compound 24 (5 nmol) was synthesized by General Procedure 2 and isolated crude after ethanol precipitation. Solid 24 was dissolved (92.5 μL) in tris buffer (50 mM) at pH 8.5. To this solution TAMRA Azide (2 μL) was added from stock solution (5 mM) in DMA, (final concentration of 100 μM). Next, BTTP (2 μL) was added from a stock solution (40 mM in water). Next, CuSCri (1 μL) was added from a stock solution (40 μM in water) (a final concentration of 400 μM). The solution was capped and vortexed. Finally, sodium ascorbate (2.5 uL) was added from a stock solution (100 mM in water). This reaction solution was vortexed and incubated for 1 hour at 37 °C. This reaction was injected onto the H-Class HPLC after 60 minutes. CuAAC product 44 was characterized below. Example - CuAAC Reaction:
Figure imgf000071_0001
[00146] Compound 24 (5 nmol) was synthesized by General Procedure 2 and isolated crude after ethanol precipitation. Solid 24 was dissolved (92.5 μL) in tris buffer (50 mM) at pH 8.5. To this solution Biotin Azide (2 μL) was added from stock solution (5 mM) in DMA, (final concentration of 100 μM). Next, BTTP (2 μL) was added from a stock solution (40 mM in water). Next, CuSCri (1 μL) was added from a stock solution (40 μM in water) (a final concentration of 400 μM). The solution was capped and vortexed. Finally, sodium ascorbate (2.5 uL) was added from a stock solution (100 mM in water). This reaction solution was vortexed and incubated for 1 hour at 37 °C. This reaction was injected onto the H-Class HPLC after 60 minutes. CuAAC product 43 was characterized below.
Example - Phosphorothioate (PS) and modified nucleotide compatibility:
[00147] Protocol for SENDR on PS DNA: SENDR phsophorothioate (PS) compatibility was analyzed by performing reactions in microcentrifuge tube as described in General Procedure 2. Briefly, 5 nmol of PS DNA 46 was loaded onto 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum.
[00148] To the loaded and dried resin, PSI-module in MeCN (150 mM, 250 μL) was added. Then DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at 37°C for 60 minutes. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of aNaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum, the DNA was dissolved, and HPLC-MS analysis was performed. Exemplary modified oligonucleotides produced by this method comprise
Figure imgf000072_0001
Example - SENDR on Aptamers:
[00149] Aptamer compatibility protocol: SENDR Aptamer compatibility was analyzed by performing reactions in microcentrifuge tube as described in General Procedure 2. Briefly, 5 nmol of aptamer 54 was loaded onto 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum.
[00150] To the loaded and dried resin, ψ-4 in MeCN (150 mM, 250 μL) was added. Then DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at 37°C for 60 minutes. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1 : 1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed. Exemplary modified oligonucleotides produced by this method comprise the following structure and SEQ ID NOs:6-7.
Figure imgf000073_0001
Example - hNE Inhibition Assay
[00151] Protocol: R/S-(57) were used directly in the assay after isolation, without further purification.
[00152] Activity of human neutrophil elastase (hNE) was measured using the method described previously2 with slight modifications in a total volume of 10 μL in a reaction buffer of PBS (pH 7.4) and 0.05% (v/v) Nonidet™P 40 Substitute (Sigma). Final composition of each reaction was 5 nM hNE (Elastin Products Corp.), 50 μM AAPV-aminomethylcoumarin (AMC) substrate (Millipore), and various concentrations of compounds. hNE was incubated with inhibitors for 10 min at room temperature before addition of AAPV-AMC. Residual proteolytic activity was measured kinetically at 25 °C using a Synergy HI microplate reader (BioTek) for a total of 30 min at 30 sec intervals (Excitation: 380 nm, Emission: 460 nm). Only data points reflecting linear substrate conversion were used to determine relative protease activity. IC50 values were obtained by fitting the data to a concentration-response inhibition, log (inhibitor) vs. response -variable slope (four parameters) using GraphPad Prism.
[00153] The same protocols were used to elicit the ability for small molecule electrophiles to inhibit hNe. All of the small molecules below proved inactive.
[00154] Small Molecule Electrophiles: The same protocols were used to elicit the ability for small molecule electrophiles to inhibit hNe. All of the small molecules below proved inactive
Figure imgf000074_0001
Examnle - SENDR on Biosvntheticallv Derived DNA
[00155] A biosynthetically derived single stranded DNA was obtained. PCR amplification was performed on this biosynthetically derived DNA.
[00156] PCR Amplification: To a PCR tube template strand (2 μL, 5 ng from 2 ng/μL stock), primer 1 (2.5 μL from a 10 μM stock), primer 2 (SI-20) (2.5 μL from a 10 μM stock), water (18 μL) were added on ice. To this mixture Q5 High Fidelity 2x Master Mix (NEB) was added (25 μL). Tubes were place into a preheated (95°C) thermocycler. An initial denaturing step of 95 °C for 30 was performed. This was followed by 30 rounds standard PCR protocol, with a denaturing step at 95 °C for 10 seconds, an annealing step at 59 °C for 15 seconds and an elongation step at 72 °C for 20 minute. A final extension step was performed at 752 °C for 2 minutes. The PCR products were purified by ZymoSpin Oligo Clean and Concentrator and eluted in 50 μL and carried forward.
[00157] Lambda Exonuclease Degradation: To the DNA mixture from the previous step (40 μL), CutSmart (5 μL, lOx), water (3 μL), and Lambda Exonuclease (2 μL) was added in a PCR tube. This mixture was vortexed and incubated at 37°C for 30 minutes at which point the lambda exonuclease was thermally denatured at 75°C for 10 minutes. The resulting ssDNA (not phosphorylated) was isolated using a ZymoSpin Oligo clean and concentrator column.
[00158] T4PNK Phosphorylation: To the DNA mixture from the previous step (30 μL), CutSmart (5 μL, 10x), DTT (5 μL, 50 mM Stock), ATP (5 μL from 10 mM stock), water (3 uL) and T4PNK (2 μL) was added in a PCR tube. This mixture was vortexed and incubated at 37°C for 60 minutes at which point the T4PNK was thermally denatured at 75°C for 10 minutes. The resulting ssDNA was isolated using a ZymoSpin Oligo clean and concentrator column. Example - DNA BSA Conjugation
[00159] DNA was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 60 was loaded onto two tubes each containing 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. These tubes were manipulated in parallel.
[00160] ψ-8 in MeCN (150 mM, 250 μL) was added to the loaded and dried resin. Then
DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (- 20°C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC- MS analysis was performed.
[00161] The DNA was dissolved to 300 μM in PBS. Solid BSA was dissolved in PBS (2.5 mg/mL, ~40 μM). these two solutions were combined in a PCR tube (25 μL of each for a reaction volume of 50 μL) and the resulting reaction mixture was incubated in a thermocycler for 4 hours. The crude ligation solution was diluted to 0.5 mg/mL with respect to BSA and injected for intact protein analysis. Deconvolution across the entire mass peak showed no detectible unmodified BSA remaining
Example - DNA-Antibodv (h38C2 IgG1) Conjugation
[00162] Procedure for the creation of SI-40: DNA SI-40 (SEQ ID NO: 8) was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 63 was loaded onto two tubes containing 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. These tubes were manipulated in parallel. [00163] ψ-5 in MeCN (150 mM, 250 μL) was added to the loaded and dried resin. Then
DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes. The reaction was worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed.
Figure imgf000076_0001
[00164] Crude isolated DNA SI-40 was purified using RP HPLC. After purification 15 nMol (15% total starting material) of pure SI-40 was obtained. To SI-40 a CuAAC reaction was performed. Compound SI-40 (15 nmol) was dissolved (92.5 μL) in tris buffer (50 mM) at pH 8.5. To this solution the b-lactam azide (2 μL) was added from stock solution (30 mM) in DMA, (final concentration of 300 μM). Next, BTTP (2 μL) was added from a stock solution
(40 mM in water). Next, CuSO4 (1 μL) was added from a stock solution (40 μM in water) (a final concentration of 400 mM). The solution was capped and vortexed. Finally, sodium ascorbate (2.5 uL) was added from a stock solution (100 mM in water). This reaction solution was vortexed and incubated for 1 hour at 37 °C. The resulting DNA 64 was ethanol precipitated, to remove excess small molecules, as previously described. Mass recovery and conversion for this transformation was assumed to be quantitative for subsequent protein conjugation.
[00165] DNA-Antibody (h38C2 IgGl) conjugation procedure: Antibody in storage solution was buffer exchanged into PBS by amicon spin filter (30 kDA) and concentrated to 25 mM (~5 mg/mL). This solution was used to dissolve (50 μL) solid DNA to a presumptive concertation of 300 mM. In addition, a second tube containing 50 μL of Antibody was aliquoted and taken through an identical process to serve as a positive control. Dissolving 64 with antibody solution was performed by vortexing the solution. This reaction mixture was incubated at 37 °C for 8 hours to produce DNA-Antibody conjugate. The crude reactions mixture was analyzed for conjugation efficiency as previously described by Rader et. al.
[00166] Methodol assay: The methodol assay for conjugation confirmation was performed as described by Rader and co-workers. Briefly, aliquots (12.5 μL) of the reaction and control solutions were diluted (0.2 mg/mL relative to original antibody concentration) in PBS to a final volume of 310 μL. Each sample were dispensed (98 μL) in triplicate into a black 96-well plate. Three blank wells, containing PBS were also dispensed (98 μL) into the black plate. A plate reader was prepared, the wavelength of excitation (λ ext) was set to 330 nm and wavelength of emission (Lem) was set to 452 nm. The instrument was programed to record every minute for 60 minutes and shake the plate in between. Finally, methodol (10 mM in ethanol) was added (2μL) to each well using a multichannel pipette and the plate was immediately loaded into the plate reader and data collection initiated. Signal was determined by normalizing against the blank wells. Measurements in triplicate were averaged and plotted along with standard deviation. Standard deviation was usually smaller than the marker size.
[00167] SDS PAGE Analysis: SDS Page was performed to confirm mass increase of the antibody-DNA construct. For this analysis 1 pg of the reaction and the control were aliquoted into PCR tubes. To these tubes 6X Lamelli buffer (6μL) was added and finally the tubes were diluted with water (to 24 μL). These reactions were heated at 95 °C for 10 minutes before being loaded into separate lanes on a precast Bio-Rad 4-20% SDS PAGE Gel. To another lane of
Bio-Rad Precsion Plus Protein standard was added (7 μL). The rest of the lanes were loaded with 6x Lamelli buffer (6μL). The gel was run at 200V for 30 minutes at which point it was stained with Coomassie protein stain and distained with water over night. Finally, the gel was imaged on a Bio-Rad gel imager.
Example - Creation of Dual Labeled Probes:
[00168] SENDR was used to access the dual label probes. The first tag was appended to the 3’ end of DNA using General Procedure 2. Briefly, 25 nmol of precursor was loaded onto 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. ψ-5 in MeCN (150 mM, 250 μL) was added to the loaded and dried resin. Then DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds and incubated at 37 °C for various amounts of time. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected by carefully pipetting the supernatant. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20°C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed.
[00169] Dephosphorylation of the 5’ end of the same DNA to generate an intermediate product DNA along with degradation of unlabeled 3’ DNA was carried out as follows. The crude reaction mixture from the previous step was dissolved in water (120 μL). This solution was aliquoted (40μL) into three separate PCR tubes. To these tubes, Cut Smart 10X Buffer was added (5μL) and the tubes were vortexed. Finally, rSAP (2.5 μL) and ExoIII (2.5 uL) was added to each tube. These tubes were vortexed, spun down and placed in a thermocycler. They were incubated at 37 °C for 60 min, then the enzymes were deactivated at 70 °C for 10 minutes. The reaction mixtures were isolated by zymo spin column.
[00170] The intermediate product DNA was loaded onto resin and modified with SENDR by General Procedure 2 using ψ-17 to generate the dual labeled DNA product. After this reaction, the crude reaction mixture is ethanol precipitated, pelleted, dried, dissolved and analyzed by HPLC-MS. [00171] Lambda Exo Cleanup: The dual labeled DNA probe was loaded into a PCR tube (5 μg) in 30 uL water. To each tube CutSmart (5 μL at 10x), ATP (5μL at 5 mM) and DTT (5μL at 50 mM) was added. Finally Lambda exonuclease (2.5 μL) and T4PNK (2.5 μL) were added. These tubes were incubated for 90 minutes at 37°C at which point the enzymes were thermally deactivated at 75°C for 10 minutes.
[00172] The dual labeled DNA was added to a PCR tube in 38 uL water. To this tube DMSO (8 uL), Tris buffer (2 μL, 1M at pH 8.5) and DBCO Fluorescein (2 μL, 10 mM, in DMSO) were added. This reaction was allowed to incubate at 37°C for 120 minutes and excess regents were removed by zymo spin column. Resulting in FAM labeled probe. To the probe in water (30 μL), Tris Buffer (1 uL, 1 M pH 8.5), BTTP (2 μL, 20 mM in water), CuS04 (1 μL, 20 mM, in water), DMSO (14 μL), MgCL2 (1 μL, 1 M in water), and BHQ-Azide (1 μL, 5 mM in DMSO) were added. Finally, NaAsc (1 μL lOOmM) was added and the reaction was incubated at 37°C for 60 minutes. The final dual labeled probe was isolated by ethanol precipitation and analyzed by HPLC MS.
[00173] An exemplary dual labeled DNA (SEQ ID NO:9) prepared by the above method is shown below:
Figure imgf000079_0001
Figure imgf000080_0001
Example -Comparison Between Kit and Microtube Format
[00174] Procedure: DNA 3 was synthesized in cartridges according to General Procedure 3. Briefly, the resin (100 μL) was loaded into a fritted cartridge (Bio-Rad, Bio-Spin column, 1.2 mL, Cat. Num. 7326008). PBS (500 μL) was added to the resin bed and allowed to flow through with gravity. The column was capped on the bottom and DNA was loaded (5 nmol in 200 μL PBS) onto the resin bed. The column was then capped (on top), vortexed and agitated for 5 minutes. The load buffer was allowed to flow out with gravity. The Resin bed was washed with DMA (500 μL twice) and THF (500 μL three times), and the solvent was allowed to flow through with gravity each time. The top cap was replaced, and the resin was dried for 2 hours under vacuum (placed on a lyophilizer). A new cap was replaced on the bottom and ψ-l was added (300 mM in MeCN, 125 μL) and then DBU was added (900 mM in MeCN, 125 μL). The top cap was replaced, the cartridge was vortexed and the column was incubated at 37 °C for 60 minutes. After the reaction the caps were removed, and the reaction mixture was allowed to flow to waste. The reaction was worked up by washing the resin with PBS and 1:1 PBS:MeCN (500 μL each). The cap washed replaced and elute buffer added (300 μL). The cartridge as vortexed for 30 seconds and agitated for 10 minutes. The cap was removed and the elute buffer was collected. Finally, the DNA was isolated by ethanol precipitation and analyzed by HPLC MS.
Example - P(V) Reagent Synthesis
Figure imgf000081_0001
[00175] To a slurry of phosphorus pentasulfide (60.0 g, 265 mmol, 1.0 equiv.) in DCM (300 mL, 5 v.) was charged pentafluorophenol (98.4 g, 530 mmol, 2.0 equiv.) under a nitrogen atmosphere. Triethylamine (78 mL, 560 mmol, 2.1 equiv.) was then added over a period of 30 minutes. Note: exothermic, maintain the reaction below 40 °C. After addition, the reaction was heated to 35 °C and held for 2-5 hours. Note: After 1 hour, the area percent ratio of product to pentafluorophenol is >50: 1. The quality of P2S5 is important to achieving excellent conversion. The reaction was cooled to ambient temperature and a mixture of MTB E/hexanes (1:1, 0.5 L, 8.3 v.) was added. The resulting mixture was washed with water (3 x 200 mL). Note: The use of hexanes alone caused the product to oil out during work up. The organic phase was concentrated to -150 mL, solids formed during concentration. Methanol (150 mL) was added and the resulting mixture was concentrated to -150 mL. Hexanes (75 mL) was added, then water (100 mL) was added slowly over 25 minutes. The resulting slurry was agitated for 1.5 hours. The batch was filtered and the reactor rinsed with methanol/water (1 : 1, 90 mL), and then hexanes (2 x 60 mL), providing PFP P(V) salt as a white solid (121 g, 81%).
Physical State: white solid;
1H NMR (400 MHz, Chloroform-d ): δ 8.79 (s, br, 1H), 3.29 (qd, J=7.3, 5.3 Hz, 6H), 1.42 (t, J= 7.3 Hz, 9H);
13C NMR (101 MHz, Chloroform-d ): δ 143.3, 140.9, 139.3, 136.8, 46.6, 8.5;
19F NMR (376 MHz, Chloroform-d ): δ -150.94 (m, 4F), -161.53 (m, 2F), -164.08 (m, 4F); 31P NMR (162 MHz, Chloroform-d ): δ 114.9 (s, IP);
HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd. for [C12F10O2PS2-H]- 460.8918; found 460.8919;
Figure imgf000082_0001
[00176] To a solution of PFP P(V) salt (50.0 g, 88.8 mmol, 1.0 equiv.) and ethylene sulfide (5.35 mL, 88.8 mmol, 1.0 equiv.) inDCM (150 mL, 3 v.) was added TFA (20.1 mL, 266 mmol, 3.0 equiv.) over 30 minutes at 0 °C. After 1 hour, ethylene sulfide (5.35 mL, 88.8 mmol, 1.0 equiv.) was added. The reaction was stirred for 18 hours at 0 °C. Hexanes (300 mL, 6v.) was added followed by water (100 mL, 2 v.). After agitating for 10-15 minutes, the aqueous layer was removed. The organic phase was washed with K3PO4 (5% aqueous, 250 mL, 5v.) and triethylamine (0.062 mL, 0.5M %). [Note: The rate of conversion and stability of the product are related to the amount of triethylamine added and the pH of the aqueous phase. More triethylamine and higher pH led to faster conversion but more decomposition, under current conditions, the decomposition of product in the biphasic mixture is <10% in 24 hours. After 45 minutes, the ratio of adduct/product was 0.12], K3PO4 (5% aqueous, 50 mL) was added, and this was agitated for 30 minutes. The aqueous layer was removed, and the organic layer was washed with H3PO4 (100 mL) followed by NaH2P04 (100 mL). The organic layer was dried over MgSCri, filtered and concentrated to -125 mL [Note: Solids formed during concentration]. This was cooled to 0 °C and stirred for 30 minutes then the product was collected by filtration. The filter cake was rinsed with cold hexanes (2 x 50 mL) and dried under vacuum affording ψ2 (24.6 g, 80%)
Physical State: white solid; 1H NMR (600 MHz, Chloroform-d ): δ 3 89 - 3 74 (m, 4H);
13C NMR (101 MHz, Chloroform-d ): δ 142.05, 142.00, 139.22, 139.18, 138.01, 137.98, 125.97, 125.84, 77.48, 77.16, 76.84, 42.42;
19F NMR (376 MHz, Chloroform-d ): δ -152.76 - -152.96 (m), -160.92 - -161.19 (m), -164.86 (t, J = 21.1 Hz);
31P NMR (162 MHz, Chloroform-d ): δ 12745;
HRMS (ESI-TOF, m/z): Calcd for [C8H4F5O2PS2+H]+ 322.9389; Found 322.9388;
Rf= 0.80 (40% EtOAc in Hexanes); UV, KMnO4
Figure imgf000083_0001
[00177] To a mixture of phosphorus pentasulfide (30.0 g, 132 mmol, 1.0 equiv.) in toluene (240 mL, 8 mL/g) was added 4-bromothiophenol (50.5 g, 267 mmol, 2.0 equiv.). The batch was made inert by flushing with nitrogen for 2 minutes. Triethylamine (39.0 mL, 277 mmol, 2.1 equiv.) was added over a period of 30 minutes (Note: The batch temperature reached 45 °C at the end of addition; the solution was allowed to reach ambient temperature by air cooling over an additional 30 minutes). The solution turned opaque yellow on addition of trimethylamine, then became cloudy. The mixture was stirred at ambient temperature overnight. The resulting slurry was filtered and the reaction vessel rinsed with toluene (2 x 30 mL); the rinses were flushed through the filter cake. The combined filtrates were concentrated under vacuum to 105 g (~3.5v). Methanol (180 mL, 6v) was added, followed by heptane (180 mL, 6v). The biphasic mixture was stirred for 15 minutes. Water (150 mL, 5v) was added over a period of 30 minutes. After the addition of water was complete, the batch was mixed for 1 hour, then filtered. The reactor was washed with water/methanol (3:2 v/v, 75 mL) and the rinse was through the filter cake. The filter cake was washed with water (2 x 90 mL) followed by heptane (2 x 45 mL). The filter cake was dried in vacuo for 15 hours at 50 °C. The product was isolated as a white crystalline solid (60.3 g, 80% yield). Physical State: white crystalline solid; 1H NMR (400 MHz, Chloroform-d ): δ 7.57 (dd, J = 8.5, 2.1 Hz, 4H), 7.52 - 7.45 (m, 4H), 3.29 - 3.19 (m, 6H), 1.38 (td, J = 7.3, 3.6 Hz, 9H);
13C NMR (151 MHz, Chloroform-d ): δ 136.46, 136.42, 132.14, 131.74, 131.71, 131.01, 46.39, 8.83;
31P NMR (162 MHz, Chloroform-d ): δ 9955;
HRMS (ESI-TOF, m/z): Calcd for [C1 2H8Na Br2S4+H]+ 470.7770; Found 470.7760.
Figure imgf000084_0001
[00178] (+)-cis-limonene oxide (50 mL) was hydrogenated with a balloon of H2 gas using Pd/C (500 mg, 10%, Johnson Matthey) as a catalyst in ethanol (200 mL). After stirring overnight at room temperature, the crude reaction was filtered through a pad of silica gel to remove the catalyst, after concentration, crude product was obtained as a clear liquid that was used directly in the next reaction.
Figure imgf000084_0002
[00179] To a solution of PBTP P(V) salt (100 g, 174.4 mmol, 1 equiv.) and (+)-hCLO (36.3 g, 235 mmol, 1.35 equiv.) in chloroform (350 mL) was added dibutylphosphate (33 mL, 174 mmol, 1 equiv.) followed by dichloroacetic acid (22 mL, 266 mmol, 1.5 equiv.). The reaction was warmed to 60 °C over 10 minutes. The reaction was aged for 1 hour at 60 °C. [Note: After 30 minutes HPLC analysis revealed the reaction was complete (crude d.r 5.75 :1)]. The crude reaction was concentrated to remove ~ 50-100 mL of solvent (Note: crystals began to form). Water (250 mL, 2.5 v.) was added followed by heptane (250 mL, 2.5 v.) and the reaction was cooled to 5 °C and stirred for 30 minutes. The crude product was collected by filtration, washed with water followed by hexane. Yielding intermediate 1 as a as a white solid (75 g, d.r >99: 1). The crude solids were mixed with DCM (400 mL) (Note: slurry with water layer on top). The water layer was discarded, and the slurry was concentrated to -100 mL. Heptane (150 mL) was added, and the slurry was concentrated to -100 mL. The product was collected by filtration and washed with a small amount of hexane yielding (54.3 g, 71% yield) as a white crystalline solid
Physical State: white crystalline solid; 1H NMR (400 MHz, Chloroform-d ): δ 7.62 - 7.46 (m, 4H), 3.53 (dt, J = 12.9, 3.4 Hz, 1H), 2.04 (ddq, J = 13.1, 3.2, 1.7 Hz, 1H), 1.85 - 1.77 (m, 1H), 1.72 - 1.56 (m, 8H), 1.47 (dd, J = 15.2, 8.3 Hz, 1H), 1.29 - 1.19 (m, 1H), 0.91 (dd, J = 12.2, 6.5 Hz, 6H);
13C NMR (100 MHz, Chloroform-d ): δ 138.01, 137.97, 132.67, 132.63, 128.33, 128.25, 125.38, 125.33, 88.36, 88.32, 64.52, 40.99, 33.04, 32.96, 27.89, 27.76, 26.60, 22.95, 22.05, 21.72, 21.53;
31P NMR (162 MHz, Chloroform-d ): δ 101 32;
HRMS (ESI-TOF, m/z): Calcd for [C16H21NaBrOPS3+H]+436.9832; Found 436.9834.
Figure imgf000085_0001
[00180] Intermediate 1 (20 g, 45.7 mmol, 1 equiv.) in DCM (300 mL, 15 v) was added SeCh (5.1 g, 45.7 mmol, 1 equiv.). The reaction was stirred for 2 hours with an overhead stirrer then SeCh (5.1 g, 45.7 mmol, 1 equiv.) was added and the reaction was stirred overnight. The mixture was filtered and washed with DCM. The filtrate was solvent swapped to heptane (480 mL, 24 v.) and cooled to 0 °C. To the organic layer was added cold NaH2PO4 (10% aqueous, 120 mL, 6v.) followed by cold bleach (8%, 120 mL, 6 v.). After mixing for 10-20 minutes (Note: The organic layer should turn colorless, otherwise perform a second wash) the organic layer was washed with NaH2PO4 (10% aqueous, 40 mL, 2 v.) and passed through a pad of MgS04. The volatiles were removed and the product slurried in heptane (80 mL, 4 v.) The product was collected by filtration and washed with heptane, yielding ψ0 as a white crystalline solid (11 g. 57% yield)
Physical State: white crystalline solid; 1H NMR (600 MHz, Chloroform-d ): δ 7.54 - 7.48 (m, 4H), 3.21 (dd, J = 13.0, 3.9 Hz, 1H), 1.95 (dq, J = 13.5, 1.9 Hz, 1H), 1.78 - 1.72 (m, 1H), 1.61 - 1.51 (m, 2H), 1.49 (td, J = 12.7, 12.0, 5.5 Hz, 1H), 1.39 (dt, J = 10.8, 5.2 Hz, 1H), 1.12 (ddd, J = 13.3, 10.9, 6.4 Hz, 1H), 0.85 (dd, J = 11.8, 6.6 Hz, 6H);
13C NMR (151 MHz, Chloroform-d ): δ 138.20, 138.17, 132.64, 132.62, 125.76, 125.71, 125.09, 125.06, 87.62, 77.37, 77.16, 76.95, 63.05, 63.04, 40.90, 33.14, 33.07, 27.49, 27.40, 26.59, 23.03, 22.19, 21.66, 21.39;
31P NMR (162 MHz, Chloroform-d ): δ 58.77;
HRMS (ESI-TOF, mix): Calcd for [C16H22BrO2PS2+H]+ 421.0060; Found 421.0070.
Example - Rac-ψ Reagent Synthesis
Figure imgf000086_0001
[00181] PFTP P(V) Salt (126 g, 0.212 mol, 1 eq.) and cyclohexene oxide (45 mL, 0.424 mol, 2 eq.) were dissolved in DCM (300 mL). To this was added TFA (32.4 mL, 0.424 mol, 2 eq.). The reaction was stirred at 25 °C overnight. The reaction was diluted with hexane (600 mL), washed successively with NaHCO3, H2O, KH2PO4 and brine. The reaction was dried over MgSCri, filtered and concentrated. The crude solid obtained was slurried with hexanes and the product was isolated as a white solid by filtration and washed with hexanes. The mother liquor was concentrated (1/2 volume) and was stored in the fridge for 1 hour. The remaining product was isolated by filtration and washed with hexanes affording Rac-ψ (77.8 g, 197 mmol, 93% yield).
Physical State: white crystalline solid;
1H NMR (600 MHz, Chloroform-d ): δ 4.14 (td, J = 10.5, 4.1 Hz, 3H), 3.70 - 3.61 (m, 1H), 3.17 (ddd, J = 12.1, 10.2, 3.7 Hz, 2H), 2.67 (s, 1H), 2.35 (dtt, J = 9.0, 3.4, 1.5 Hz, 3H), 2.22 (dddd, J = 14.5, 5.4, 3.7, 1.8 Hz, 1H), 2.18 - 2.14 (m, 1H), 2.16 - 2.07 (m, 2H), 2.00 - 1.90 (m, 2H), 1.88 (dt, J = 14.9, 2.0 Hz, 1H), 1.88 - 1.81 (m, 2H), 1.77 - 1.65 (m, 2H), 1.67 - 1.56 (m, 3H), 1.55 - 1.41 (m, 5H), 1.43 - 1.35 (m, 1H), 1.35 - 1.24 (m, 3H), 1.26 - 1.18 (m, 1H).; 13C NMR (100 MHz, Chloroform-d ): δ 148.48, 148.25, 148.20, 148.02, 147.97, 143.18, 143.14, 143.11, 143.07, 142.07, 137.95, 137.91, 137.87, 137.80, 106.56, 105.30, 105.23, 104.71, 104.64, 88.88, 88.84, 87.73, 87.69, 73.08, 58.66, 57.71, 57.34, 34.22, 32.45, 31.28, 31.15, 30.89, 30.74, 30.40, 30.31, 29.73, 29.63, 26.13, 25.29, 25.25, 24.26, 24.12, 24.06;
19F NMR (376 MHz, Chloroform-d ): δ -129.80 - -133.43 (m), -149.09 - -153.89 (m), -161.01 - -164.00 (m);
31P NMR (162 MHz, Chloroform-d ): δ 105 3, 994;
HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for [C12H10F5O2PS2+H]+ 376.9858; Found 376.9859.
Example - Selected Experimental Data:
Figure imgf000087_0001
[00182] Prepared according to General Procedure 1 using 2-(2-(2- azidoethoxy)ethoxy)ethan-1-ol (0.70 g, 4.0 mmol). Purification using a gradient of 50-100% DCM in hexanes followed by 0-10% EtOAc in DCM afforded ψ-3 (0.809 g, 1.92 mmol, 48% yield).
Physical State: Colorless oil;
Rf= 0.70 (100% DCM);
1H NMR (400 MHz, Chloroform- d): δ 4.98 (s, 1H), 4.87 (s, 1H), 4.44 (dd, J = 12.8, 3.3 Hz,
1H), 4.41 - 4.32 (m, 1H), 4.32 - 4.20 (m, 1H), 3.80 - 3.60 (m, 9H), 3.39 (t, J = 4.1 Hz, 2H), 2.57 (s, 1H), 2.27 (d, J = 13.6 Hz, 1H), 2.21 - 2.02 (m, 1H), 2.00 - 1.83 (m, 3H), 1.78 (s, 4H), 1.69 (d, J = 2.7 Hz, 3H);
13C NMR (151 MHz, Chloroform-d ): δ 145.25, 111.86, 85.83, 70.83, 70.79, 70.26, 70.21, 70.17, 67.67, 67.62, 65.20, 50.83, 39.00, 33.86, 33.80, 27.90, 27.79, 23.50, 22.81, 21.80;
31P NMR (162 MHz, Chloroform-d ): δ 102 15;
HRMS (ESI-TOF, m/z): Calcd for [C16H28N3O4PS2+H]+ 422.1337; Found 422.1327.
Figure imgf000088_0001
[00183] Prepared according to General Procedure 1 using 4-pentynol (1.26 g, 15.0 mmol). Purification using a gradient of 15-20% DCM in hexanes afforded ψ-5 (4.56 g, 13.8 mmol, 92%).
Physical State: Colorless Oil;
Rf= 0.46 (50 % DCM in Hexanes);
1H NMR (600 MHz, Chloroform-d ): δ 5.01 (q, J = 1.5 Hz, 1H), 4.90 - 4.86 (m, 1H), 4.44 (dt, J = 12.7, 3.3 Hz, 1H), 4.32 - 4.25 (m, 1H), 4.27 - 4.20 (m, 1H), 2.58 (t, J = 6.4 Hz, 1H), 2.30 (qd, J = 8.4, 7.7, 2.3 Hz, 3H), 2.11 (td, J = 13.5, 4.3 Hz, 1H), 1.99 - 1.84 (m, 6H), 1.80 - 1.72 (m, 4H), 1.70 (s, 3H);
13C NMR (151 MHz, Chloroform-d ): δ 144.63, 111.32, 85.22, 82.34, 68.73, 66.76 (d, J = 7.9 Hz), 64.87, 38.44, 33.34 (d, J = 9.0 Hz), 28.54 (d, J = 7.5 Hz), 27.27 (d, J = 15.5 Hz), 22.97, 22.20, 21.24, 14.49;
31P NMR (162 MHz, Chloroform-d ): δ 101.32;
[α]D 20 = -20 (c 2.0, CHCl3 );
HRMS (ESI-TOF, m/z): Calcd for [C15H23O2PS2+H]+ 331.0955; Found 331.0963.
Figure imgf000088_0002
[00184] Prepared according to General Procedure 1 using SI-51 (0.90 g, 3.0 mmol). Purification using 25% EtOAc in hexanes afforded ψ-7 (1.0 g, 1.82 mmol, 61% yield).
Physical State: colorless solid;
Rf= 0.28 (30% EtOAc/Hexane);
1H NMR (400 MHz, Chloroform-d ): 8.17 - 8.11 (m, 1H), 7.90 - 7.84 (m, 1H), 7.80 - 7.72 (m, 2H), 5.25 (s, 1H), 4.99 (s, 1H), 4.86 (s, 1H), 4.45 - 4.37 (m, 1H), 4.13 - 4.10 (m, 2H), 3.10 (q, J = 6.8 Hz, 2H), 2.58 (s, 1H), 2.27 (d, J = 13.5 Hz, 1H), 2.15 - 2.05 (m, 1H), 1.90 (td, J = 21.4, 18.7, 14.3 Hz, 4H), 1.78 (s, 3H), 1.69 (d, J = 2.0 Hz, 3H), 1.62 (d, J = 6.5 Hz, 2H), 1.53 (d, J = 7.0 Hz, 2H), 1.33 (s, 4H); 13C NMR (151 MHz, Chloroform-d ): 148.22, 145.25, 133.84, 133.73, 132.95, 131.21, 125.54,
111.83, 85.79, 68.72, 68.67, 65.41, 43.85, 39.00, 33.92, 33.86, 29.99, 29.95, 29.58, 27.91,
27.80, 26.08, 25.13, 23.52, 22.79, 21.80;
31P NMR (162 MHz, Chloroform-d ): δ 100.65;
HRMS (ESI-TOF, m/z): Calcd for [C22H33N2O6PS3+H]+ 549.1317; Found 549.1326,
Figure imgf000089_0001
[00185] Prepared according to General Procedure 1 using SI-50 (1.35 g, 4.67 mmol). Purification using 100% DCM afforded ψ-8 (1.54 g, 3.15 mmol, 68% yield).
Physical State: Light Yellow Oil;
Rf= 0.50 (100% DCM);
1H NMR (600 MHz, Chloroform-d ): δ 8.45 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 7.71 (dt, J = 8.1, 1.1 Hz, 1H), 7.64 (td, J = 7.7, 1.8 Hz, 1H), 7.08 (ddd, J = 7.4, 4.8, 1.1 Hz, 1H), 4.98 (q, J = 1.4 Hz, 1H), 4.87 - 4.84 (m, 1H), 4.42 (dt, J = 12.8, 3.3 Hz, 1H), 4.19 - 4.07 (m, 2H), 2.79 (t, J = 7.3 Hz, 2H), 2.57 (s, 1H), 2.27 (ddt, J = 15.0, 3.2, 1.7 Hz, 1H), 2.09 (td, J = 13.5, 4.2 Hz, 1H), 1.98 - 1.91 (m, 1H), 1.91 - 1.82 (m, 2H), 1.79 - 1.73 (m, 4H), 1.73 - 1.63 (m, 7H), 1.42 (pd, J = 7.1, 3.0 Hz, 2H), 1.41 - 1.31 (m, 2H);
13C NMR (151 MHz, Chloroform-d ): δ 160.67, 149.73, 145.22, 137.10, 120.67, 119.72, 111.86, 85.71, 68.86 (d, J = 8.2 Hz), 65.35, 38.96 (d, J = 14.8 Hz), 33.90 (d, J = 8.8 Hz), 30.06 (d, J = 6.8 Hz), 28.86, 28.07, 27.91, 27.81, 25.30, 23.54, 22.81, 21.80;
31P NMR (162 MHz, Chloroform-d ): δ 101.01;
HRMS (ESI-TOF, m/z): Calcd for [C21H32NO2PS4+H]+ 490.1132; Found 490.1140.
Figure imgf000090_0001
[00186] Prepared according to General Procedure 1 using SI-56 (0.368 g, 1.0 mmol). Purification using a gradient of 20-50% EtOAc in hexanes afforded ψ-10 (0.382 g, 0.62 mmol, 62% yield).
Physical State: dark orange foam;
Rf= 0.58 (50% EtO Ac/Hexane);
1H NMR (400 MHz, Chloroform-d ): δ 8.06 - 7.68 (m, 6H), 6.76 (d, J = 8.6 Hz, 2H), 6.19 (s, 1H), 5.00 (s, 1H), 4.87 (s, 1H), 4.44 (d, J = 12.9 Hz, 1H), 4.17 (t, J = 7.9 Hz, 2H), 3.48 (d, J = 6.9 Hz, 3H), 3.11 (p, J = 1.9 Hz, 6H), 2.57 (s, 1H), 2.29 (s, 1H), 2.16 - 2.03 (m, 1H), 1.92 (dd, J = 25.4, 14.3 Hz, 2H), 1.71 (dd, J = 32.2, 18.3 Hz, 10H), 1.45 (s, 5H);
13C NMR (151 MHz, Chloroform-d ): δ 167.17, 154.94, 152.98, 145.23, 143.73, 134.92, 127.90, 125.67, 122.34, 111.89, 111.73, 85.78, 68.90, 68.85, 65.38, 40.50, 40.13, 39.02, 33.93, 33.88, 30.11, 30.07, 29.73, 27.93, 27.82, 26.58, 25.39, 23.54, 22.81, 21.81;
31P NMR (162 MHz, Chloroform-d ): δ 101.78;
HRMS (ESI-TOF, m/z): Calcd for [C31H43N4 O3PS2+H]+ 615.2592; Found 615.2593.
Figure imgf000090_0002
[00187] Prepared according to General Procedure 1 using SI-57 (0.292 g, 1.0 mmol). Purification using a gradient of 75-100% EtOAc in hexanes afforded ψ-11 (0.35 g, 0.65 mmol, 65% yield).
Physical State: Amourphous solid;
Rf= 0.32 (100% EtOAc);
1H NMR (400 MHz, Chloroform-d ): δ 8.52 (s, 1H), 7.94 (d, J = 9.0 Hz, 1H), 7.17 (d, J = 8.9 Hz, 1H), 6.24 (s, 1H), 4.97 (s, 1H), 4.84 (s, 1H), 4.40 (d, J= 12.8 Hz, 1H), 4.13 -4.09 (m, 2H), 3.40 (q, J = 7.0 Hz, 2H), 2.55 (s, 1H), 2.25 (d, J = 13.6 Hz, 1H), 2.04 (s, 3H), 1.87 (d, J = 15.8 Hz, 6H), 1.71 (d, J = 34.1 Hz, 8H), 1.59 (s, 2H), 1.39 (d, J = 6.5 Hz, 4H) ;
13C NMR (151 MHz, Chloroform-d ): δ 165.64, 158.20, 152.28, 148.76, 147.96, 146.17, 145.22, 137.73, 135.23, 123.65, 121.76, 111.88, 107.05, 85.78, 68.90, 68.84, 65.38, 39.97, 39.02, 33.93, 33.87, 30.09, 30.05, 29.73, 27.92, 27.82, 26.54, 25.48, 25.35, 23.54, 22.81, 21.81, 16.36;
31P NMR (162 MHz, Chloroform-d ): δ 101.18;
HRMS (ESI-TOF, m/z): Calcd for [C25H39N4O3PS2+H]+ 539.2279; Found 539.2280.
Figure imgf000091_0001
[00188] To a solution of (-)-ψ (0.447 g, 1.0 mmol, 1 equiv.) in THF (10 mL) was added phenylethynylmagnesium bromide (2.0 mL, 2.0 mmol, 2.0 equiv., 1.0 m solution, THF). After stirring for 2 hours the reaction was diluted with 1 :1 Et20/hexanes (50 mL) and washed consecutively with water, KH2PO4 (saturated aqueous) and brine. The organic layer was dried over MgSCri, filtered and concentrated to a yellow oil. Purification in 10% Et20 in hexanes afforded ψ-15 (0.25 g, 0.72 mmol, 72% yield).
Physical State: yellow amorphous solid;
Rf= 0.4 (10% Et20/hexanes);
1H NMR (600 MHz, Chloroform-d ): δ 7.54 (dd, J= 7.1, 2.0 Hz, 2H), 7.44 (td, J= 7.4, 1.9 Hz, 1H), 7.37 (td, J= 7.8, 2.0 Hz, 2H), 5.02 - 4.97 (m, 1H), 4.91 (s, 1H), 4.56 (ddt, J= 12.8, 6.1, 2.8 Hz, 1H), 2.61 (d, J= 6.4 Hz, 1H), 2.41 - 2.35 (m, 1H), 2.19 - 2.08 (m, 1H), 1.97 (td, J= 11.0, 9.3, 3.4 Hz, 3H), 1.83 - 1.75 (m, 1H), 1.80 (s, 3H), 1.71 (s, 1H);
13C NMR (151 MHz, Chloroform-d ): δ 145.29, 132.59, 132.57, 130.84, 128.64, 119.88, 119.85, 111.85, 101.85, 101.60, 86.73, 86.62, 85.33, 64.71, 38.98, 33.99, 33.93, 28.36, 28.26, 23.63, 22.84, 22.54;
31P NMR (162 MHz, Chloroform-d ): δ 61.01;
HRMS (ESI-TOF, m/z): Calcd for [C18H21OPS2+H]+ 349.0850; Found 349.0844.
Figure imgf000092_0001
[00189] Prepared according to General Procedure 1 using SI-58 (1.4 g, 5.24 mmol). Purified in 30-50% EtOAc in hexanes (2.3 g, 4.48 mmol, 85% yield).
Physical State: white foam;
Rf= 0.66 (60% EtO Ac/Hexanes);
1H NMR (600 MHz, Chloroform-d ): δ 8.34 (s, 1H), 7.36 (q, J= 1.3 Hz, 1H), 6.35 (dd, J = 8.6, 5.7 Hz, 1H), 5.26 (ddt, J= 11.5, 6.7, 2.4 Hz, 1H), 5.06 (q, J= 1.4 Hz, 1H), 4.92 - 4.88 (m, 1H), 4.45 (dt, J= 12.7, 3.2 Hz, 1H), 4.26 (q, J= 3.0 Hz, 1H), 3.80 - 3.70 (m, 2H), 2.60 (t, J= 6.1 Hz, 1H), 2.49 (ddd, J= 14.2, 5.7, 2.1 Hz, 1H), 2.35 - 2.23 (m, 2H), 2.13 (td, J= 13.5, 4.2 Hz, 1H), 1.98 - 1.85 (m, 6H), 1.80 (s, 3H), 1.80 - 1.72 (m, 1H), 1.70 (s, 3H);
13C NMR (151 MHz, Chloroform-d ): δ 162.71, 149.64, 144.28, 134.36, 111.67, 111.36, 85.74, 83.88, 82.59, 82.55, 77.86, 77.81, 65.68, 51.75, 38.36, 38.16, 38.13, 33.27, 33.21, 27.31, 27.21, 22.93, 22.20, 21.32, 12.26;
31P NMR (162 MHz, Chloroform-d ): δ 10222 ;
HRMS (ESI-TOF, m/z): Calcd for [C20H28N5O5PS2+H]+ 514.1348; Found 514.1350
Figure imgf000092_0002
[00190] Prepared according to General Procedure 1 using SI-59 (1.4 g, 5.00 mmol). Purified in 50% EtOAc in hexanes (2.13 g, 4.05 mmol, 83% yield).
Physical State: white foam;
Rf= 0.27 (50% EtOAc/Hexanes);
1H NMR (600 MHz, Chloroform-d ): δ 8.30 (s, 1H), 7.44 (q, J= 1.2 Hz, 1H), 6.30 (dd, J = 8.4, 5.7 Hz, 1H), 4.97 (q, J= 1.5 Hz, 1H), 4.83 (dd, .7= 2.1, 1.1 Hz, 1H), 4.51 (ddd, J= 12.5, 11.5, 3.4 Hz, 1H), 4.43 (ddd, J= 12.8, 3.6, 2.5 Hz, 1H), 4.40 - 4.30 (m, 2H), 4.28 - 4.23 (m, 1H), 4.23 - 4.18 (m, 1H), 4.21 - 4.09 (m, 1H), 2.60 (s, 1H), 2.51 - 2.44 (m, 2H), 2.28 (ddt, J = 13.4, 3.2, 1.7 Hz, 1H), 2.08 (td, J= 13.4, 4.2 Hz, 1H), 2.04 (s, 1H), 2.01 - 1.93 (m, 5H), 1.95 - 1.90 (m, 1H), 1.92 - 1.85 (m, 1H), 1.82 - 1.72 (m, 7H);
13C NMR (151 MHz, Chloroform-d ): δ 163.60, 150.04, 144.52, 134.86, 111.34, 110.95, 85.99, 84.42, 82.32, 82.27, 78.52, 78.01, 75.10, 67.08, 67.02, 65.50, 56.49, 38.34, 36.93, 33.31, 33.26, 27.27, 27.17, 22.85, 22.14, 21.23, 12.31;
31P NMR (162 MHz, Chloroform-d ): δ 102.69;
HRMS (ESI-TOF, m/z): Calcd for [C23H31N2O6PS2+H]+ 527.1439; Found 527.1431
Figure imgf000093_0001
[00191] To a solution of adamantane methanol (1.0 equiv.) and PSI(O) (1.5 equiv.) in DCM (0.1 M) was added DBU (1.5 equiv.). After 1.5 hours the reaction was diluted with EtOAc and washed with NaHCO3, water, KH2PO4 and brine. The organic layer was dried over MgSCri filtered and concentrated. The crude product was purified via column chromatography or non- polar compounds such as this can be recrystallized from MeOH.
Physical State: white crystalline solid;
31P NMR (162 MHz, Chloroform-d ): δ 42.2 (s) ;
Figure imgf000093_0002
[00192] ψ 2-dG was prepared according to General Procedure 1 using dG(Pya) (SI-7) (5.00 g, 7.69 mmol). Purification by SFC (Waters Torus DIOL column 5 mm, 19x160 mm, 100 mL/min, 22-30% MeOH in CO2 over 3 min 120 bar backpressure at 40 °C) afforded compoundψ 2 -dG (0.800 g, 13%).
Physical State: white foam;
1H NMR (600 MHz, Acetonitrile-*): δ 9.46 (s, 1H), 7.69 (s, 1H), 7.40 - 7.35 (m, 2H), 7.25 (ddt, J = 9.8, 3.9, 1.9 Hz, 6H), 7.22 - 7.17 (m, 1H), 6.84 - 6.77 (m, 4H), 6.24 (t, J= 6.7 Hz, 1H), 5.38 (ddt, J= 14.2, 7.2, 3.7 Hz, 1H), 4.26 (dt, .7= 5.5, 3.9 Hz, 1H), 3.76 - 3.59 (m, 10H), 3.62 - 3.53 (m, 1H), 3.47 - 3.36 (m, 2H), 3.31 (dd, J= 10.5, 4.0 Hz, 1H), 3.26 (dd, J= 10.6, 5.4 Hz, 1H), 3.08 - 2.92 (m, 4H), 2.98 (s, 3H), 2.73 - 2.67 (m, 1H), 2.00 - 1.91 (m, 1H);
13C NMR (151 MHz, Acetonitrile-*): δ 170.21, 159.62, 159.60, 158.81, 157.64, 151.12, 145.83, 137.56, 136.62, 136.55, 130.97, 130.90, 128.91, 128.78, 127.80, 120.99, 114.01, 87.23, 84.93, 84.86, 84.81, 79.38, 79.32, 64.12, 55.87, 51.94, 42.96, 42.90, 38.49, 38.46, 32.02, 31.95, 20.29;
31P NMR (162 MHz, Acetonitrile-*): δ 122.94;
HRMS (ESI-TOF, m/z): HRMS (ESI) Calcd for [C38H41N5O6PS3+H]+ 805.2066; Found 805.2061.
Examnle - DNA BSA Conjugation
Procedure for the creation of 61
[00193] DNA 61 was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 60 was loaded onto two tubes each containing 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. These tubes were manipulated in parallel.
[00194] ψ-8 in MeCN (150 mM, 250 μL) was added to the loaded and dried resin. Then
DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes. The reactions were worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (- 20°C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC- MS analysis was performed (Fig. 10).
Procedure for the creation of 62
[00195] Compound 61 was synthesized by General Procedure 2 and isolated crude after ethanol precipitation. DNA 61 was dissolved to 300 mM in PBS. Solid BSA was dissolved in PBS (2.5 mg/mL, ~40 μM). these two solutions were combined in a PCR tube (25 μL of each for a reaction volume of 50 μL) and the resulting reaction mixture was incubated in a thermocycler for 4 hours. The crude ligation solution was diluted to 0.5 mg/mL with respect to BSA and injected for intact protein analysis. Deconvolution across the entire mass peak showed no detectible unmodified BSA remaining. (Fig. 11)
Procedure for the creation of SI-40
Figure imgf000095_0001
[00196] DNA SI-40 was synthesized using General Procedure 2. Briefly, 100 nmol of DNA 63 was loaded onto two tubes containing 100 μL of equilibrated resin. This loaded resin was washed with DMA (500 μL twice) and dry THF (500 μL three times) and dried under vacuum. These tubes were manipulated in parallel.
[00197] ψ-5 in MeCN (150 mM, 250 μL) was added to the loaded and dried resin. Then
DBU (450 mM, 18 μL) was added to the reaction mixture. The reaction tube was vortexed for 30 seconds incubated at 37 °C for 60 minutes. The reaction was worked up by aspirating and discarding the reaction solution, and washing the resin bed with MeCN (500 μL) and 1:1 MeCN:PBS (500 μL). Elute buffer (300 μL) was added to the resin bed and the tube was agitated by orbital shaker for 5-10 minutes. The DNA containing elution buffer was collected. DNA was isolate via ethanol precipitation. Ethanol precipitation was performed by adding 10% v/v of a NaCl solution (30 μL, 5M) to the elute buffer and three volumes of cold ethanol (-20 °C) were added to the tube (~1000μL) and incubated for 18 hours at -20 °C. The tube was then centrifuged at 13,000 rpm for 15 minutes to pellet the DNA, and the extra ethanol was decanted off. The tubes were dried via speed vacuum and the DNA was dissolved and HPLC-MS analysis was performed. (Fig. 12)
Procedure for the creation 64
Figure imgf000096_0001
[00198] Crude isolated DNA SI-40 was purified using RP HPLC. After purification 15 nMol (15% total starting material) of pure SI-40 was obtained. To SI-40 a CuAAC reaction was performed. Compound SI-40 (15 nmol) was dissolved (92.5 μL) in tris buffer (50 mM) at pH 8.5. To this solution the b-lactam azide SI-53 (2 μL) was added from stock solution (30 mM) in DMA, (final concentration of 300 μM). Next, BTTP (2 μL) was added from a stock solution (40 mM in water). Next, CuSCfi (1 μL) was added from a stock solution (40 μM in water) (a final concentration of 400 μM). The solution was capped and vortexed. Finally, sodium ascorbate (2.5 uL) was added from a stock solution (100 mM in water). This reaction solution was vortexed and incubated for 1 hour at 37 °C. DNA 64 was ethanol precipitated, to remove excess small molecules, as previously described. Mass recovery and conversion for this transformation was assumed to be quantitative for subsequent protein conjugation. (Fig. 13).
DNA-Antibodv (h38C2 IgGl) conjugation procedure
[00199] Antibody (h38C2 IgGl) in storage solution was buffer exchanged into PBS by amicon spin filter (30 kDA) and concentrated to 25 μM (~5 mg/mL). This solution was used to dissolve (50 μL) solid DNA 64 to a presumptive concertation of 300 μM. In addition, a second tube containing 50 μL of Antibody was aliquoted and taken through an identical process to serve as a positive control. Dissolving 64 with antibody solution was performed by vortexing the solution. This reaction mixture was incubated at 37 °C for 8 hours to produce DNA- Antibody conjugate 65. The crude reactions mixture was analyzed for conjugation efficiency as previously described by Rader et. al 3
Methodol Assay
[00200] The methodol assay for conjugation confirmation was performed as described by Rader and co-workers. Briefly, aliquots (12.5 μL) of the reaction and control solutions were diluted (0.2 mg/mL relative to original antibody concentration) in PBS to a final volume of 310 μL. Each sample were dispensed (98 μL) in triplicate into a black 96-well plate. Three blank wells, containing PBS were also dispensed (98 μL) into the black plate. A plate reader was prepared, the wavelength of excitation (λext) was set to 330 nm and wavelength of emission (λem ) was set to 452 nm. The instrument was programed to record every minute for 60 minutes and shake the plate in between. Finally, methodol (10 mM in ethanol) was added (2μL) to each well using a multichannel pipette and the plate was immediately loaded into the plate reader and data collection initiated. Signal was determined by normalizing against the blank wells. Measurements in triplicate were averaged and plohed along with standard deviation. Standard deviation was usually smaller than the marker size. Protocol adapted from: A. R. Nanna and C. Rader Methods Mol Biol, 2019, 2033, 39-52. (Fig. 14)
SDS PAGE Analysis
[00201] SDS Page was performed to confirm mass increase of the antibody-DNA construct (Fig. 15). For this analysis 1 μg of the reaction and the control were aliquoted into PCR tubes. To these tubes 6X Lamelli buffer (6μL) was added and finally the tubes were diluted with water (to 24 μL). These reactions were heated at 95 °C for 10 minutes before being loaded into separate lanes on a precast Bio-Rad 4-20% SDS PAGE Gel. To another lane of Bio-Rad Precsion Plus Protein standard was added (7 μL). The rest of the lanes were loaded with 6x Lamelli buffer (6μL). The gel was run at 200V for 30 minutes at which point it was stained with Coomassie protein stain and distained with water over night. Finally, the gel was imaged on a Bio-Rad gel imager. Protocol adapted from: A. R. Nanna and C. Rader , Methods Mol Biol, 2019, 2033, 39-52.
[00202] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the full scope of the present disclosure, and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.
[00203] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the full scope of the concepts disclosed herein. The disclosed subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refer to at least one of something selected from the group consisting of A, B, C ... . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMS What is claimed is:
1. A method for site selective chemical modification of an oligonucleotide comprising: immobilizing the oligonucleotide on a solid support to form oligonucleotide-resin complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-resin complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the solid support.
2. The method of claim 1, wherein the site-selective chemical modification of the oligonucleotide comprises modification at 5’ hydroxyl group or 3’ hydroxyl group, or both 5’ and 3’ hydroxyl groups.
3. The method of claim 1, wherein the phosphorus (V) reagent has a chemical structure
Figure imgf000099_0001
wherein X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’,
R’ is at each occurrence are independently selected from the group consisting hydrogen, C1to C10 alkyl, C1to C10 heteroalkyl, C1to C10 cycloalkyl, C1to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl;
Y and Z are each independently selected from the group consisting of O, S, and N;
R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein: any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle.
4. The method of claim 2, wherein the phosphorus (V) reagent has the chemical structure
Figure imgf000100_0001
5. The method of any one of the preceding claims, wherein the P(V) module is created in-situ.
6. The method of any one of the preceding claims, wherein the oligonucleotide a DNA, RNA, LNA, MPO, PMO, or derivations and chimeric mixtures thereof.
7. The method of any one of the preceding claims, wherein the modified oligonucleotide is modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group.
8. The method of any one of the preceding claims, wherein the oligonucleotide is a structured aptamer, primer, hybridization probe or library thereof.
9. The method of any one of the preceding claims, wherein the oligonucleotide is further conjugated to a protein to form a oligonucleotide-protein conjugate.
10. The method of claim 9, wherein the protein is a therapeutic antibody.
11. The method of any one of the preceding claims, wherein the oligonucleotide is modified at multiple specific locations.
12. The method of any one of the preceding claims, further comprising eluting the modified oligonucleotide from the resin by washing the oligonucleotide-resin complex with an aqueous buffer solution.
13. The method of any one of the preceding claims, wherein the P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes.
14. The method of any one of the preceding claims, wherein the modified oligonucleotide further conjugates with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins.
15. The method of any one of the preceding claims, wherein the biorthogonal chemistry comprises Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffinity labeling, hydrazone ligation, and CuAAC reaction.
16. The method of any one of the preceding claims, wherein the oligonucleotide comprises unnatural nucleotides.
17. The method of claim 15, wherein the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
18. A method of increasing the half-life of an oligonucleotide therapeutic in-vivo, the method comprising: immobilizing the oligonucleotide on an inert resin to form oligonucleotide-resin complex; reacting a phosphorus (V) reagent with a nucleophile to generate a P(V) module; contacting the oligonucleotide-resin complex with the P(V) module in an organic solvent to produce a modified oligonucleotide; and eluting the modified oligonucleotide from the inert resin, wherein the site specific P(V) reagent modification increases the half-life of the oligonucleotide therapeutic in vivo.
19. The method of claim 18, wherein the site-selective chemical modification of the oligonucleotide comprises modification at 5’ hydroxyl group, or 3’ hydroxyl group or both 5’ and 3’ hydroxyl groups.
20. The method of claim 18, wherein the phosphorus (V) reagent has a chemical structure
Figure imgf000102_0001
wherein X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’,
R’ is at each occurrence are independently selected from the group consisting hydrogen, C1to C10 alkyl, C1to C10 heteroalkyl, C1to C10 cycloalkyl, C1to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl Y and Z are each independently selected from the group consisting of O, S, and N
R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein: any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle.
21. The method of claim 18, wherein the phosphorus (V) reagent has the chemical structure
Figure imgf000103_0001
22. The method of any one of claims 18-21, wherein the P(V) module is created in-situ.
23. The method of any one of claims 18-22, wherein the oligonucleotide a DNA, RNA, LNA, MPO, PMO, or derivations or chimeric mixtures thereof.
24. The method of any one of the preceding claims, wherein the modified oligonucleotide is modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group.
25. The method of any one of claims 18-24, wherein the oligonucleotide is a structured aptamer or hybridization probe.
26. The method of any one of claims 18-25, wherein the oligonucleotide is further conjugated to a protein to form an oligonucleotide-protein conjugate.
27. The method of claim 26, wherein the protein is a therapeutic antibody.
28. The method of any one of claims 18-27, wherein the oligonucleotide is modified at multiple specific locations.
29. The method of any one of claims 18-28, further comprising eluting the modified oligonucleotide from the resin by washing the oligonucleotide-resin complex with an aqueous buffer solution.
30. The method of any one of claims 18-29, wherein the P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes.
31. The method of any one of claims 18-20, wherein the modified oligonucleotide further conjugates with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins.
32. The method of claim 31, wherein the biorthogonal chemistry comprises Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffmity labeling, hydrazone ligation, and CuAAC reaction.
33. The method of any one of claims 18-32, wherein the oligonucleotide comprises unnatural nucleotides.
34. The method of claim 33, wherein the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
35. A method of targeting delivery of an oligonucleotide therapeutic to a specific location in the patient body, the method comprising: attaching a sugar or lipid to a therapeutic oligonucleotide by the method of claim 1 to thereby produce a modified therapeutic oligonucleotide; and administering the modified therapeutic oligonucleotide to the patient for targeted delivery to a specific location in the patient body.
36. The method of claim 35, wherein the site-selective chemical modification of the oligonucleotide comprises modification at 5’ hydroxyl group, or 3’ hydroxyl group or both 5’ and 3’ hydroxyl groups.
37. The method of claim 35, wherein the phosphorus (V) reagent has a chemical structure
Figure imgf000105_0001
wherein X is a leaving group having the formula O-R’, S-R’, N-R’, C-R’,
R’ is at each occurrence are independently selected from the group consisting hydrogen, C1to C10 alkyl, C1to C10 heteroalkyl, C1to C10 cycloalkyl, C1to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl;
Y and Z are each independently selected from the group consisting of O, S, and N;
R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide, amino, sugar, carbohydrate, and lipid, wherein:
38. any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle
39. The method of any one of the preceding claims, wherein the P(V) module is created in-situ.
40. The method of any one of claims 35-39, wherein the oligonucleotide therapeutic a DNA, RNA, LNA, MPO, PMO, or derivations or chimeric mixtures thereof.
41. The method of any one of claims 35-40, wherein the modified therapeutic oligonucleotide is modified in the phosphate linkages, in the base, in the backbone, or in the 2’ group.
42. The method of any one of claims 35-41, wherein the therapeutic oligonucleotide is a structured aptamer or hybridization probe.
43. The method of any one of claims 35-42, wherein the therapeutic oligonucleotide is further conjugated to a protein to form a oligonucleotide-protein conjugate.
44. The method of claim 43, wherein the protein is a therapeutic antibody.
45. The method of any one of claims 35-44, wherein the oligonucleotide is modified at multiple specific locations.
46. The method of any one of claims 35-45, further comprising eluting the modified oligonucleotide from the resin by washing the oligonucleotide-resin complex with an aqueous buffer solution.
47. The method of any one of claims 35-46, wherein the P(V) reagent comprises small molecule reactive handles, affinity tags, fluorophores, and FMRI probes.
48. The method of any one of claims 35-47, wherein the modified oligonucleotide further conjugates with chemical or biological entities through bioorthogonal chemistry, to create oligonucleotide conjugated to small molecules, peptides, and proteins.
49. The method of claim 48, wherein the biorthogonal chemistry comprises Strain Promoted Azide Alkyne Cycloaddition (SPAAC), Inverse Electron Demand Diels-Alder (IEDDA), unsymmetrical disulfide Formation, amide coupling, proximity photoaffmity labeling, hydrazone ligation, and CuAAC reaction.
50. The method of any one of claims 35-49, wherein the oligonucleotide comprises unnatural nucleotides.
51. The method of claim 50, wherein the unnatural nucleotides comprise polymerase incompetent nucleosides, Locked Nucleic Acids (LNA), or epigenetic modified nucleosides.
52. A kit for DNA modification, comprising: a. A P(V) reagent having the formula
Figure imgf000107_0001
wherein X= O-R’, S-R’, N-R’, C-R’,
R’ is at each occurrence are independently selected from the group consisting hydrogen, C1 to C10 alkyl, C1 to C10 heteroalkyl, C1 to C10 cycloalkyl, C1 to C10 heterocycle, C1 to C10 aryl, C1 to C10 heteroaryl and C1 to C10 aralkyl;
Y and Z are each independently selected from the group consisting of O, S, and N;
R1, R2, R3, and R4 are each independently selected from the group consisting of hydrogen, — OH, — CN, — NO2, halogen, C1 to C20 alkyl, C1 to C20 heteroalkyl, cycloalkyl, heterocycle, aryl, heteroaryl, aralkyl, alkoxy, alkoxy carbonyl, alkanoyl, carbamoyl, substituted sulfonyl, sulfonate, sulfonamide and amino, wherein: any two of R1, R2, R3, and R4, either both on a single C or on adjoining C's, together with the C or C's to which they are attached, optionally form a cycle, b. 1,8-Diazabicyclo[5.4. 0]undec-7-ene (DBU) c. Dry Tetrahydrofuran (THF) d. Dimethylacetamide (DMA) e. Elution buffer f. a solid support in fitted cartridge
53. The kit of claim 52, wherein the elution buffer comprises 1M NaC1O4, 20% MeOH, 40 mM Tris pH 8.5.
54. The kit of claim 52, wherein the solid support is Strata AXL resin.
55. A method of diagnosing a disease in a patient in need thereof, comprising: obtaining a tissue sample, blood sample, or body fluid swab from the patient; extracting oligonucleotides from the patient sample; labeling and/or modifying the oligonucleotides that would be cognate to disease of interest by the method of claim 1; performing PCR reaction on the labeled and/or modified oligonucleotide; and analyzing the labeled and/or modified oligonucleotide to diagnose the disease.
56. The method of claim 55, wherein the disease is an infectious disease
57. The method of claim 55, wherein the disease is a bacterial or viral disease, and the patient is diagnosed with the disease if the analyzed labeled and/or modified oligonucleotide comprises the bacterial or viral DNA.
58. The method of claim 55, wherein the disease is a tumor, and the patient is diagnosed with the tumor if the analyzed labeled and/or modified oligonucleotide comprises cancer specific DNA mutations.
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Publication number Priority date Publication date Assignee Title
WO2024131790A1 (en) * 2022-12-20 2024-06-27 Nanjing University A bioorthogonal cycloaddition reaction and use thereof
CN117700468A (en) * 2024-02-05 2024-03-15 深圳赛陆医疗科技有限公司 Preparation method of nucleotide analogue
CN117700468B (en) * 2024-02-05 2024-05-03 深圳赛陆医疗科技有限公司 Preparation method of nucleotide analogue

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